NOEY2 gene compositions and methods of use

ABSTRACT

Disclosed are compositions and methods comprising a novel tumor suppressor gene, designated NOEY2, that is expressed in normal ovarian and breast surface epithelial cells but consistently absent or down-regulated in ovarian and breast cancer cells. Disclosed are polynucleotide compositions comprising a NOEY2 gene from mammalian sources, and polypeptides encoded by these nucleic acid sequences. Also disclosed are methods for preparing NOEY2 polypeptides, transformed host cells, and antibodies reactive with NOEY2 polypeptides. In certain embodiments, the invention describes methods for diagnosing and treating cancers, as well as methods for identifying NOEY2-related polynucleotide and polypeptide compositions.

The present application is a continuing application of U.S. ProvisionalApplication Ser. No. 60/071,263 filed Jan. 13, 1998, which is acontinuing application of U.S. Provisional Application Ser. No.60/041,580 filed Mar. 21, 1997; the entire contents of each beingspecifically incorporated herein by reference in its entirety.

The United States government has rights in the present inventionpursuant to Grant Number CA39930 from the National Cancer Institute.

1.0 BACKGROUND OF THE INVENTION

1.1 Field of the Invention

The present invention relates generally to the fields of oncology andmolecular biology. More particularly, it concerns nucleic acid segmentsisolated from human chromosome 1p31, which encode a novel tumorsuppressor protein, designated NOEY2. Various methods for making andusing NOEY2 DNA segments, DNA segments encoding synthetically-modifiedNOEY2 proteins, and native and synthetic tumor suppressor proteins aredisclosed, such as, for example, the use of DNA segments as diagnosticprobes and templates for protein production, and the use of proteins,fusion protein carriers and peptides in various immunological anddiagnostic applications. Also disclosed are methods for identifyingNOEY2-related tumor suppressor polynucleotides and polypeptides, andmethods for treating tumors, and in particular, ovarian andbreast-related cancers.

1.2 Description of Related Art

Oncogenesis was described by Foulds (1958) as a multistep biologicalprocess, which is presently known to occur by the accumulation ofgenetic damage. On a molecular level, the multistep process oftumorigenesis involves the disruption of both positive and negativeregulatory effectors (Weinberg, 1989). Defects leading to thedevelopment of retinoblastoma have been linked to a tumor suppressorgene (Lee et al., 1987), and a variety of oncogenes and other tumorsuppressors have been identified in a host of malignancies.Unfortunately, there remains an inadequate number of treatable cancers,and the effects of cancer are catastrophic—over half a million deathsper year in the United States alone.

1.2.1 Tumor Suppressors

Tumor suppressor proteins function to negatively regulate cell cycleprocesses, preventing the uncontrolled growth exhibited by cancerouscells. Tumor suppressor proteins function by transcriptional regulationof key genes involved in cellular growth and division. Unlikeprotooncogenes, for which activation is required for the initiation ofcancerous growth, inactivation of tumor suppressors leads to cancer. Innormal diploid cells, two copies of the gene encoding a particular tumorsuppressor protein are present. The “two hit” hypothesis states thatmutation or inactivation of both individual copies of the gene isrequired for the onset of cancerous growth. Mutations may includedeletions, alterations to transcription levels, single or multiplecoding changes, and truncations. Inherited mutations in a single tumorsuppressor gene typically leads to a recessive heterozygous phenotype.Cells remain phenotypically normal until mutation of the second, wildtype copy of the gene. Familial inheritance of a mutant copy of a tumorsuppressor gene does render the individual more prone to cancer, asinactivation of the single normal gene is sufficient to initiate cancer.The two most heavily studied tumor suppressor genes are theretinoblastoma gene Rb and the p53 gene.

The retinoblastoma gene Rb was the first discovered tumor suppressorgene. The Rb protein is a 110 kDa nuclear phosphoprotein that reducesthe growth rate of cells, and mutations in this protein have been foundin breast, prostate, and small cell lung carcinomas. The Rb proteinbinds to DNA, but without any sequence specificity. Rb effects itsregulatory function through the formation of complexes withtranscription factors. Rb has been shown to interact with transcriptionfactor E2F, which binds to the promoters of cellular genes such as DNApolymerase α and ribonucleotide reductase. Heterozygous individualsinherit one defective and one normal copy of the Rb gene. Uponinactivation of the second, functional copy of the gene, cancerousgrowth initiates. A wide range of mutations including deletions,duplications, and point mutations have been found to lead toinactivation of the Rb protein.

The p53 tumor suppressor has been found to be mutated in about 60% ofhuman cancerous growths, making p53 the most commonly mutated gene inhuman cancers. Wild type p53 protein binds to DNA and is believed tofunction as a transcriptional regulator. The p53 protein also binds tothe mdm2 protein, commonly expressed at high levels in tumors. Levels ofwild type p53 protein increases upon subjecting a cell to radiation orchemical agents which damage DNA. The p53 protein has been implicated inDNA repair mechanisms that prevent the duplication of damaged or alteredDNA. It has been further speculated that p53 protein prevents the cellfrom entering S phase until the complete repair of damaged DNA has beenachieved. The p53 protein is normally found at low, often undetectablelevels in cells, and has a short cellular half life. Mutant p53 proteinsoften are more resistant to degradation, and are present atimmunohistochemically detectable levels in cancerous cells. Most p53gene mutations occur in four highly conserved regions. Mutant p53proteins are dominant inactivators, by their ability to bind to andinactivate wild type p53 protein.

1.2.2 Ovarian Cancer

The ovary is the fifth most common site of cancer among American womenand ovarian neoplasms constitute the fourth leading cause of cancerdeath. Ovarian cancer affects 22,000 women in the United States eachyear and causes some 14,000 deaths annually. Approximately 90% ofovarian cancers arise from epithelial cells that cover the ovariansurface or that line inclusion cysts. Ovarian cancers exhibit adistinctive pattern of metastasis. Like other epithelial neoplasms,ovarian cancer can metastasize to pelvic and retroperitoneal lymph nodesand can spread hematogenously to distant sites. More frequently,however, ovarian cancer cells spread over the surface of the peritonealcavity, forming multiple nodules on the parietal and visceralperitoneum. Blockade of diaphragmatic lymphatics and increasedtransudation of fluid produce accumulation of ascites fluid thatcontains varying numbers of tumor cells. Abdominal distention bymalignant ascites is a frequent mode of clinical presentation. Earlystage ovarian cancer rarely produces symptoms and at present there is noproven strategy for early detection. In more than 75% of cases, tumorcells have metastasized beyond the ovaries at presentation. Initialclinical management generally involves cytoreductive surgery anddrainage of ascites, providing large amounts of tissue for study.Following removal of as much tumor as possible, cytotoxic chemotherapyis generally administered. Introduction of platinum based compounds andthe taxane derivatives has improved median survival of patients withadvanced disease, but the five year survival rate is still only 28% forall stages and has not improved in the last several decades. Diseasefrequently persists and recurs within the peritoneal cavity, producingintestinal obstruction. Therapeutic strategies that prevent suchprogression and that treat disease regionally by intraperitonealinfusion continue to hold promise. Identification of genes whoseaberrant function can be demonstrated in ovarian carcinomas may haveimportant diagnostic and therapeutic applications.

1.2.3 Breast Cancer

Breast cancer is the most common form of cancer among women, affectingabout one in eight women. Approximately 185,700 new cases are diagnosedin the U.S. annually, and breast cancer is responsible for about 44,560deaths in the U.S. per year. While predominantly observed in women,1,400 cases of breast cancer are diagnosed annually in men, and 260 mendie of breast cancer per year. Breast cancer first manifests itself as apainless lump, detectable by self-examination and clinical breast examsincluding mammograms. Commonly, growth initiates in the lining of theducts or in the lobules of the breast. Current clinical treatmentsinclude mastectomy (removal of the entire breast) or lumpectomy (removalof the tumor and surrounding tissue) for localized tumors. Chemotherapy,radiotherapy, or hormone-blocking therapy may be further used to controlcancerous cells. Breast cancer cells can metastasize to the lymph nodes,skin, lungs, liver, brain, or bones. Metastasis may occur early or latein the disease progression, although typically metastasis occurs oncethe cancerous growth reaches a size of about 20 mm. Metastasis isachieved by cells breaking away from the parental mass and enteringeither the bloodstream or the lymphatic system.

Genetic inheritance appears to play a role in about 5–10% of breastcancer patients. Mutations in the BRCA1, BRCA2, and p53 tumor suppressorgenes have been observed to confer high risks of breast and ovariancancers. BRCA1 mutations are present at between 1 in 300 to 1 in 800females. In the BRCA1 gene, over 200 different mutations have beendiscovered to date. The mutations observed are not localized to a singleregion, further complicating genetic analysis. Greater than 80% of theobserved mutations result in a truncated form of the BRCA1 protein.Individuals with familial hereditary BRCA1 possess one normal and onemutant form of the gene, and are therefore much more likely to developbreast cancer. It is estimated that women with a hereditary BRCA1mutation are about 76% likely to develop breast cancer by 70 years ofage.

BRCA2 has been identified on chromosome 13q through linkage analysis of15 breast cancer families that did not demonstrate BRCA1 linked breastcancer. Unlike BRCA1 mutations, BRCA2 does not substantially elevate therisk of ovarian cancers. The BRCA2 gene encodes a protein of 3,418 aminoacids, many of which are acidic or basic. Most mutations observedinvolve base deletions that alter the reading frame, and result in apremature truncation of the protein. BRCA1 and BRCA2 account for about45% of familial inherited breast cancers each, leaving 10% for one ormore additional genes. Interestingly, all male breast cancers appear tobe due to mutations in the BRCA2 gene.

Mutations found in breast tumor p53 genes are commonly single base pairchanges which result in variants with increased cellular half lives.Altered p53 proteins have been observed in 20–25% of breast cancers.

1.3 Deficiencies in the Prior Art

There are relatively few tumor suppressor genes whose mutations havebeen shown to correlate with the presence of cancerous cells. Those thathave been characterized typically have many possible types and positionsof mutations, complicating genetic analyses and the prediction of cancerpredisposition. Therefore, what is lacking in the prior art is theidentification and characterization of novel tumor suppressor genes, andidentification of the role of the proteins encoded by such genes incancer diagnosis and treatment. Such genes, in combination with improvedgenetic testing, and improved correlation of specific genetic mutationswith particular cancer susceptibility are needed to facilitate early andeffective treatment of these proliferative diseases.

2.0 SUMMARY OF THE INVENTION

The present invention overcomes these and other limitations in the priorart by providing a novel tumor suppressor protein (designated NOEY2)(SEQ ID NO:2) and the gene which encodes it (designated NOEY2). Thisgene is expressed in normal ovarian surface epithelial cells, butconsistently absent or down-regulated in ovarian cancer cells. Theinvention provides unique polynucleotide sequences which comprise thecDNA (SEQ ID NO:1) and the genomic DNA (SEQ ID NO:5) encoding this NOEY2tumor suppressor. A further objective of the invention is to providepolynucleotide segments comprising all or parts of a gene encodingNOEY2. Polynucleotide probes and primers specific for these NOEY2 genesalso represent important compositions provided by the invention. It is afurther objective of the invention to provide antibodies specific forNOEY2, methods for identifying NOEY polypeptide and polynucleotidecompositions, methods for producing such compositions, and methods forusing these compositions in a variety of diagnostic and therapeuticregimens. The invention also provides methods and compositions for thedetection of NOEY2 compositions in biological and clinical samples, andmethods for regulating the proliferation of tumor cells in vitro and invivo.

In one important embodiment, the invention provides an isolated andpurified amino acid segment comprising a NOEY2 tumor suppressor protein(SEQ ID NO:2) comprising the amino acid sequence of SEQ ID NO:2. Thecoding region for the NOEY2 polypeptide is from nucleotide 150 to 833 ofSEQ ID NO:1, (the cDNA for NOEY2). The genomic DNA sequence is presentedin SEQ ID NO:5. The NOEY2 protein exhibits tumor suppressor activitywhich is related to cyclin D1 promoter inhibition. In relatedembodiments, methods for making and using this protein, derivatives andmutants thereof, and antibodies directed against these proteins are alsodisclosed.

In another important embodiment, the invention provides an isolated andpurified nucleic acid segment comprising the NOEY2 gene which encodesthe NOEY2 tumor suppressor protein disclosed herein. The nucleotidesequence of an exemplary NOEY2 gene is given from position 150 toposition 833 of SEQ ID NO:1 (NOEY2 cDNA) and SEQ ID NO:5 (NOEY2genomic). In related embodiments, methods for making, using, altering,mutagenizing, assaying, and quantitating these nucleic acid segments arealso disclosed. Also disclosed are diagnostic methods and assay kits forthe identification and detection of related NOEY2 gene sequences in avariety of in vitro and in vivo methodologies.

Another aspect of the present invention is an animal cell, such as ahuman or other animal cell, that comprises a NOEY2 polypeptide orpolynucleotide. In a preferred embodiment, the cell is an ovariansurface epithelial cell that produces a tumor suppressor polypeptide ofapproximately 26-kDa, and that is identical to, or substantiallyhomologous with, the NOEY2 polypeptide identified in SEQ ID NO:2.

A further aspect of the present invention is a vector (such as aplasmid, cosmid, virus, phagemid, or the like), that includes within itsnucleotide sequence a nucleic acid segment that comprises one or moreNOEY2 genes, or portions thereof. Preferably such a vector is comprisedwithin a transformed host cell. The transformed host cell may be abacterial, animal, fungal, or plant cell, and may be comprised within atransgenic animal, or may be comprised within a culture of bacteria,yeast, fungus, animal or plant cells.

In another embodiment, there is provided a monoclonal antibody thatbinds immunologically to a tumor suppressor designated as NOEY2. Theantibody may be non-cross reactive with other human polypeptides, or itmay bind to non-human NOEY2, but not to human NOEY2. The antibody mayfurther comprise a detectable label, such as a fluorescent label, achemiluminescent label, a radiolabel or an enzyme. Also encompassed arehybridoma cells and cell lines producing such antibodies.

In another embodiment, there is included a polyclonal antisera,antibodies of which bind immunologically to a tumor suppressordesignated as NOEY2. The antisera may be derived from any animal, butpreferably is from an animal other than a human. Preferred antigens forthe preparation of such sera include a NOEY2 polypeptide isolated from ahuman, rat, goat, rabbit, pig, horse, cat, dog, hamster, monkey or othersuch animal cell line. Preferred hosts for the preparation of apolyclonal antisera specific for NOEY2 include animals such as rabbits,goats, and other such animals.

The invention also provides pharmaceutical compositions which compriseone or more of the NOEY2 compositions disclosed herein. Suchcompositions may include NOEY2 or NOEY2-derived polypeptides,polynucleotides, antibodies, antisera, antigens, peptide epitopes,protein fusions, peptides and the like.

In still yet another embodiment, there is provided a method ofdiagnosing a cancer comprising the steps of (a) obtaining a sample froma subject; and (b) determining the expression a functional NOEY2 tumorsuppressor in cells of the sample. Preferably the cancer is ovarian orbreast cancer, although the cancer may also be brain, lung, liver,splenic, renal, lymphatic, intestinal, pancreatic, leukemia, colon,stomach, endometrial, prostate, testicular, skin, head and neck,esophageal, bone marrow or blood cancer. In a preferred embodiment, thecancer is ovarian cancer or breast cancer. The sample is a cell, cellculture, tissue or fluid sample, and may be of clinical or non-clinicalorigin.

In one format, the method involves assaying for a nucleic acid from thesample. The method may further comprise subjecting the sample toconditions suitable to amplify the nucleic acid. Alternatively, themethod may comprise contacting the sample with an antibody that bindsimmunologically to a NOEY2, for example, in an ELISA. The comparison,regardless of format, may include comparing the expression of NOEY2 withthe expression of NOEY2 in non-cancer samples, for example in normalovarian epithelial cells. The comparison may look at levels of NOEY2expression. Alternatively, the comparison may involve evaluating thestructure of the NOEY2 gene, protein or transcript. Such formats mayinclude sequencing, wild-type oligonucleotide hybridization, mutantoligonucleotide hybridization, SSCP, PCR™ and/or RNase protection.Particular embodiments include evaluating wild-type or mutantoligonucleotide hybridization where the oligonucleotide is configured inan array, on a chip, on a wafer, or in a microtiter dish.

In another embodiment, there is provided a method for altering thephenotype of a tumor cell comprising the step of contacting the cellwith a tumor suppressor designated NOEY2 under conditions permitting theuptake of the tumor suppressor by the tumor cell. The tumor cell may bederived from an animal organ or tissue such as brain, lung, liver,spleen, kidney, lymph node, small intestine, blood cells, pancreas,colon, stomach, breast, endometrium, prostate, testicle, ovary, skin,head and neck, esophagus, or bone marrow. Preferably the tumor cell isderived from an ovarian or breast cancer cell. The phenotype may beselected from proliferation, migration, contact inhibition, soft agargrowth or cell cycling. The tumor suppressor polypeptide may be providedin a pharmaceutical formulation, encapsulated in a liposome, nanocapsuleor other lipid particle, or may be carrier-free.

In another embodiment, there is provided a method for altering thephenotype of a tumor cell comprising the step of contacting the cellwith a nucleic acid (a) encoding a NOEY2 tumor suppressor and (b) apromoter active in the tumor cell, wherein the promoter is operablylinked to the region encoding the tumor suppressor, under conditionspermitting the uptake of the nucleic acid by the tumor cell. Thephenotype may be proliferation, migration, contact inhibition, soft agargrowth or cell cycling. The tumor suppressor-encoding nucleic acid maybe provided in a pharmaceutical formulation, encapsulated in a liposome,nanocapsule or other lipid particle, or may be carrier-free. If thenucleic acid is comprised within a viral vector such as retrovirus,adenovirus, adeno-associated virus, vaccinia virus and herpesvirus, itmay also be encapsulated in a viral particle.

The invention further provides a method of inhibiting cellularproliferation, comprising providing to a cell a composition comprising aNOEY2 tumor suppressor polypeptide, or a NOEY2 polynucleotide whichexpresses a NOEY2 polypeptide in a pharmaceutically acceptable vehicle.

The present invention also provides a method of inhibiting tumorproliferation, comprising providing to a tumor cell a compositioncomprising a NOEY2 tumor suppressor polypeptide or a NOEY2 gene whichexpresses the NOEY2 protein in a pharmaceutically acceptable vehicle.

In a further embodiment, there is provided a method for treating cancercomprising the step of contacting a tumor cell within a subject with atumor suppressor designated NOEY2 under conditions permitting the uptakeof the tumor suppressor by the tumor cell. The method may involve ahuman subject.

In still a further embodiment, there is provided a method for treatingcancer comprising the step of contacting a tumor cell within a subjectwith a nucleic acid (a) encoding a NOEY2 tumor suppressor and (b) apromoter active in the tumor cell, wherein the promoter is operativelylinked to the region encoding the tumor suppressor, under conditionspermitting the uptake of the nucleic acid by the tumor cell. The subjectis preferably an animal, and most preferably a human.

In still yet a further embodiment, there is provided transgenic mammalin which both copies of the native NOEY2 gene are interrupted orreplaced with another gene.

In an additional embodiment, there is provided a method of determiningthe stage of cancer comprising the steps of (a) obtaining a sample froma subject; and (b) determining the expression a functional NOEY2polypeptide in cells of the sample. The cancer is preferably a breastcancer or ovarian cancer. The determining may comprise assaying for aNOEY2 nucleic acid or polypeptide in the sample.

In yet an additional example, there is provided a method of predictingtumor metastasis comprising the steps of (a) obtaining a sample from asubject; and (b) determining the expression a functional NOEY2polypeptide in cells of the sample. The cancer may be distinguished asmetastatic and non-metastatic. The determining may comprise assaying fora NOEY2 nucleic acid or NOEY2 polypeptide in the sample.

In still yet an additional embodiment, there is provided a method ofscreening a candidate substance for anti-tumor activity comprising thesteps of (a) providing a cell lacking functional NOEY2 polypeptide; (b)contacting the cell with the candidate substance; and (c) determiningthe effect of the candidate substance on the cell. The cell may be atumor cell, for example, a tumor cell having a mutation in the codingregion of NOEY2. The mutation may be a deletion mutant, an insertionmutant, a frameshift mutant, a nonsense mutant, a missense mutant orsplice mutant. The determining may comprise comparing one or morecharacteristics of the cell in the presence of the candidate substancewith characteristics of a cell in the absence of the candidatesubstance. The characteristic may be NOEY2 expression, phosphataseactivity, proliferation, metastasis, contact inhibition, soft agargrowth, cell cycle regulation, tumor formation, tumor progression andtissue invasion. The candidate substance may be a chemotherapeutic orradiotherapeutic agent or be selected from a small molecule library. Thecell may be contacted in vitro or in vivo.

The foregoing objects of the invention and others that are now readilyapparent to those of skill in the art having the benefit of the presentdisclosure are described more fully in the sections which follow:

2.1 NOEY2 DNA Segments

In one embodiment, the present invention concerns DNA segments, that canbe isolated from virtually any source, that are free from total genomicDNA and that encode the whole or a portion of the novel peptidedisclosed herein. The NOEY2 gene (position 150 to position 833 of SEQ IDNO:1 and SEQ ID NO:5) encodes a NOEY2 polypeptide having the contiguousamino acid sequence shown in SEQ ID NO:2. The inventors contemplate avariety of NOEY2 DNA segments from the present invention will findparticular utility. For example, those segments that encode all orportions of the NOEY2 polypeptide, or subunits, functional domains, andthe like of NOEY2 and NOEY2-related polypeptides, or those segments thatcomprise one or more NOEY2 promoter or enhancer regions will be usefulin a variety of diagnostic, and therapeutic regimens. Such DNA segmentsmay be native DNA segments isolated using molecular biological methods,or alternatively, such segments may be mutagenized segments, or evensegments which have been synthesized in vitro either partially orentirely, using chemical synthesis methods that are well-known to thoseof skill in the art.

As used herein, the term “DNA segment” refers to a DNA molecule that hasbeen isolated free of total genomic DNA of a particular species.Therefore, a DNA segment encoding a tumor suppressor protein or peptiderefers to a DNA segment that contains a NOEY2 polypeptide-codingsequence yet is isolated away from, or purified free from, total genomicDNA of the species from which the DNA segment is obtained. Includedwithin the term “DNA segment”, are DNA segments comprising entire NOEY2genes and/or promoter regions, as well as all partial and smallerfragments and subfragments isolatable from such entire gene-comprisingsegments, and also recombinant vectors (such as plasmids, cosmids,phagemids, phage, viruses, and the like) which comprise one or more ofthe NOEY2-specific polynucleotide sequences of the invention. Likewise,the segments may comprise gene sequences which are identical to, orsubstantially homologous with, a contiguous nucleotide sequence fromabout position position 150 to about position 833 of SEQ ID NO:1 or SEQID NO:5, or gene sequences which encode polypeptides which are identicalto, or substantially biologically-functionally equivalent to, thepolypeptide disclosed in SEQ ID NO:2.

Similarly, a DNA segment comprising an isolated or purified tumorsuppressor protein-encoding gene refers to a DNA segment which mayinclude in addition to peptide encoding sequences, certain otherelements such as, regulatory sequences, isolated substantially away fromother naturally occurring genes or protein-encoding sequences. In thisrespect, the term “gene” is used for simplicity to refer to a functionalprotein-, polypeptide- or peptide-encoding unit. As will be understoodby those in the art, this functional term includes not only genomicsequences, including extrachromosomal DNA sequences, but also operonsequences and/or engineered gene segments that express, or may beadapted to express, proteins, polypeptides or peptides.

“Isolated substantially away from other coding sequences” means that thegene of interest, in this case, a NOEY2 tumor suppressor gene, forms thesignificant part of the coding region of the DNA segment, and that theDNA segment does not contain large portions of naturally-occurringcoding DNA, such as large chromosomal fragments or other functionalgenes or operon coding regions. Of course, this refers to the DNAsegment as originally isolated, and does not exclude genes, recombinantgenes, synthetic linkers, or coding regions later added to the segmentby the hand of man.

In particular embodiments, the invention concerns isolated DNA segmentsand recombinant vectors incorporating DNA sequences that encode a NOEY2polypeptide that includes within its amino acid sequence an at least tenamino acid contiguous sequence from SEQ ID NO:2, and more preferablystill, a polypeptide that includes within its amino acid sequence asequence essentially as set forth in SEQ ID NO:2. In a preferredembodiment, such a DNA segment comprises a gene encoding the amino acidsequence of SEQ ID NO:2, and more preferably still, comprises apolynucleotide which is identical to, or substantially homologous with,the DNA sequence of SEQ ID NO:1 or SEQ ID NO:5.

The term “a sequence essentially as set forth in SEQ ID NO:2,” meansthat the sequence substantially corresponds to a portion of the sequenceof SEQ ID NO:2 and has relatively few amino acids that are not identicalto, or a biologically functional equivalent of, the amino acids of anyof these sequences. The term “biologically functional equivalent” iswell understood in the art and is further defined in detail herein(e.g., see Illustrative Embodiments). Accordingly, sequences that havebetween about 70% and about 80%, or more preferably between about 81%and about 90%, or even more preferably between about 91% and about 99%amino acid sequence identity or functional equivalence to the aminoacids of SEQ ID NO:2 will be sequences that are “essentially as setforth in SEQ ID NO:2.”

It will also be understood that amino acid and nucleic acid sequencesmay include additional residues, such as additional N- or C-terminalamino acids or 5′ or 3′ sequences, and yet still be essentially as setforth in one of the sequences disclosed herein, so long as the sequencemeets the criteria set forth above, including the maintenance ofbiological protein activity where protein expression is concerned. Theaddition of terminal sequences particularly applies to nucleic acidsequences that may, for example, include various non-coding sequencesflanking either of the 5′ or 3′ portions of the coding region or mayinclude various internal sequences, i.e., introns, which are known tooccur within genes.

The nucleic acid segments of the present invention, regardless of thelength of the coding sequence itself, may be combined with other DNAsequences, such as promoters, polyadenylation signals, additionalrestriction enzyme sites, multiple cloning sites, other coding segments,and the like, such that their overall length may vary considerably. Itis therefore contemplated that a nucleic acid fragment of almost anylength may be employed, with the total length preferably being limitedby the ease of preparation and use in the intended recombinant DNAprotocol. For example, nucleic acid fragments may be prepared thatinclude a short contiguous stretch encoding the whole or a portion ofthe peptide sequence disclosed in SEQ ID NO:2, or that are identical toor complementary to DNA sequences which encode the peptide disclosed inSEQ ID NO:2, and particularly the DNA segment disclosed in either of SEQID NO:1 or SEQ ID NO:5. For example, DNA sequences such as about 14nucleotides, and that are up to about 10,000, about 5,000, about 3,000,about 2,000, about 1,000, about 500, about 200, about 100, about 50, andabout 14 base pairs in length (including all intermediate lengths) arealso contemplated to be useful.

It will be readily understood that “intermediate lengths”, in thesecontexts, means any length between the quoted ranges, such as 14, 15,16, 17, 18, 19, 20, etc.; 21, 22, 23, etc.; 30, 31, 32, etc.; 50, 51,52, 53, etc.; 100, 101, 102, 103, etc.; 150, 151, 152, 153, etc.;including all integers through the 200–500; 500–1,000; 1,000–2,000;2,000–3,000; 3,000–5,000; and up to and including sequences of about10,000 nucleotides and the like.

It will also be understood that this invention is not limited to theparticular nucleic acid sequences which encode peptides of the presentinvention, or which encode the amino acid sequence of SEQ ID NO:2,including the DNA sequence which is particularly disclosed in SEQ IDNO:1 and SEQ ID NO:5. Recombinant vectors and isolated DNA segments maytherefore variously include the peptide-coding regions themselves,coding regions bearing selected alterations or modifications in thebasic coding region, or they may encode larger polypeptides thatnevertheless include these peptide-coding regions or may encodebiologically functional equivalent proteins or peptides that havevariant amino acids sequences.

The DNA segments of the present invention encompassbiologically-functional, equivalent peptides. Such sequences may ariseas a consequence of codon redundancy and functional equivalency that areknown to occur naturally within nucleic acid sequences and the proteinsthus encoded. Alternatively, functionally-equivalent proteins orpeptides may be created via the application of recombinant DNAtechnology, in which changes in the protein structure may be engineered,based on considerations of the properties of the amino acids beingexchanged. Changes designed by man may be introduced through theapplication of site-directed mutagenesis techniques, e.g., to introduceimprovements to the antigenicity of the protein or to test mutants inorder to examine activity at the molecular level.

If desired, one may also prepare fusion proteins and peptides, e.g.,where the peptide-coding regions are aligned within the same expressionunit with other proteins or peptides having desired functions, such asfor purification or immunodetection purposes (e.g., proteins that may bepurified by affinity chromatography and enzyme label coding regions,respectively).

Recombinant vectors form further aspects of the present invention.Particularly useful vectors are contemplated to be those vectors inwhich the coding portion of the DNA segment, whether encoding a fulllength protein or smaller peptide, is positioned under the control of apromoter. The promoter may be in the form of the promoter that isnaturally associated with a gene encoding peptides of the presentinvention, as may be obtained by isolating the 5′ non-coding sequenceslocated upstream of the coding segment or exon, for example, usingrecombinant cloning and/or PCR™ technology, in connection with thecompositions disclosed herein.

2.2 DNA Segments as Hybridization Probes and Primers

In addition to their use in directing the expression of the gene productof the novel tumor suppressor gene of the present invention, the nucleicacid sequences contemplated herein also have a variety of other uses.For example, they also have utility as probes or primers in nucleic acidhybridization embodiments. As such, it is contemplated that nucleic acidsegments that comprise a sequence region that consists of at least a 14nucleotide long contiguous sequence that has the same sequence as, or iscomplementary to, a 14 nucleotide long contiguous DNA segment of SEQ IDNO:1 or SEQ ID NO:5 will find particular utility. Longer contiguousidentical or complementary sequences, e.g., those of about 20, 30, 40,50, 100, 200, 500, 1000, 2000, 5000, 10000 etc. (including allintermediate lengths and up to and including full-length sequences) willalso be of use in certain embodiments.

The ability of such nucleic acid probes to specifically hybridize totumor suppressor protein-encoding sequences will enable them to be ofuse in detecting the presence of complementary sequences in a givensample. However, other uses are envisioned, including the use of thesequence information for the preparation of mutant species primers, orprimers for use in preparing other genetic constructions.

Nucleic acid molecules having sequence regions consisting of contiguousnucleotide stretches of 10–14, 15–20, 30, 50, or even of 100–200nucleotides or so, identical or complementary to the DNA sequence of SEQID NO:1 or SEQ ID NO:5, are particularly contemplated as hybridizationprobes for use in, e.g., Southern and Northern blotting. Smallerfragments will generally find use in hybridization embodiments, whereinthe length of the contiguous complementary region may be varied, such asbetween about 10–14 and about 100 or 200 nucleotides, but largercontiguous complementarity stretches may be used, according to thelength complementary sequences one wishes to detect.

The use of a hybridization probe of about 14 nucleotides in lengthallows the formation of a duplex molecule that is both stable andselective. Molecules having contiguous complementary sequences overstretches greater than 14 bases in length are generally preferred,though, in order to increase stability and selectivity of the hybrid,and thereby improve the quality and degree of specific hybrid moleculesobtained. One will generally prefer to design nucleic acid moleculeshaving gene-complementary stretches of 15 to 20 contiguous nucleotides,or even longer where desired.

Of course, fragments may also be obtained by other techniques such as,e.g., by mechanical shearing or by restriction enzyme digestion. Smallnucleic acid segments or fragments may be readily prepared by, forexample, directly synthesizing the fragment by chemical means, as iscommonly practiced using an automated oligonucleotide synthesizer. Also,fragments may be obtained by application of nucleic acid reproductiontechnology, such as the PCR™ technology of U.S. Pat. Nos. 4,683,195 and4,683,202 (each incorporated herein by reference), by introducingselected sequences into recombinant vectors for recombinant production,and by other recombinant DNA techniques generally known to those ofskill in the art of molecular biology.

Accordingly, the nucleotide sequences of the invention may be used fortheir ability to selectively form duplex molecules with complementarystretches of DNA fragments. Depending on the application envisioned, onewill desire to employ varying conditions of hybridization to achievevarying degrees of selectivity of probe towards target sequence. Forapplications requiring high selectivity, one will typically desire toemploy relatively stringent conditions to form the hybrids, e.g., onewill select relatively low salt and/or high temperature conditions, suchas provided by about 0.02 M to about 0.15 M NaCl at temperatures ofabout 50° C. to about 70° C. Such selective conditions tolerate little,if any, mismatch between the probe and the template or target strand,and would be particularly suitable for isolating tumor suppressorprotein-encoding DNA segments. Detection of DNA segments viahybridization is well-known to those of skill in the art, and theteachings of U.S. Pat. Nos. 4,965,188 and 5,176,995 (each incorporatedherein by reference) are exemplary of the methods of hybridizationanalyses. Teachings such as those found in the texts of Maloy et al.,1994; Segal 1976; Prokop, 1991; and Kuby, 1991, are particularlyrelevant.

Of course, for some applications, for example, where one desires toprepare mutants employing a mutant primer strand hybridized to anunderlying template or where one seeks to isolate tumor suppressorprotein-encoding sequences from related species, functional equivalents,or the like, less stringent hybridization conditions will typically beneeded in order to allow formation of the heteroduplex. In thesecircumstances, one may desire to employ conditions such as about 0.15 Mto about 0.9 M salt, at temperatures ranging from about 20° C. to about55° C. Cross-hybridizing species can thereby be readily identified aspositively hybridizing signals with respect to control hybridizations.In any case, it is generally appreciated that conditions can be renderedmore stringent by the addition of increasing amounts of formamide, whichserves to destabilize the hybrid duplex in the same manner as increasedtemperature. Thus, hybridization conditions can be readily manipulated,and thus will generally be a method of choice depending on the desiredresults.

In certain embodiments, it will be advantageous to employ nucleic acidsequences of the present invention in combination with an appropriatemeans, such as a label, for determining hybridization. A wide variety ofappropriate indicator means are known in the art, including fluorescent,radioactive, enzymatic or other ligands, such as avidin/biotin, whichare capable of giving a detectable signal. In preferred embodiments, onewill likely desire to employ a fluorescent label or an enzyme tag, suchas urease, alkaline phosphatase or peroxidase, instead of radioactive orother environmental undesirable reagents. In the case of enzyme tags,calorimetric indicator substrates are known that can be employed toprovide a means visible to the human eye or spectrophotometrically, toidentify specific hybridization with complementary nucleicacid-containing samples.

In general, it is envisioned that the hybridization probes describedherein will be useful both as reagents in solution hybridization as wellas in embodiments employing a solid phase. In embodiments involving asolid phase, the test DNA (or RNA) is adsorbed or otherwise affixed to aselected matrix or surface. This fixed, single-stranded nucleic acid isthen subjected to specific hybridization with selected probes underdesired conditions. The selected conditions will depend on theparticular circumstances based on the particular criteria required(depending, for example, on the G+C content, type of target nucleicacid, source of nucleic acid, size of hybridization probe, etc.).Following washing of the hybridized surface so as to removenonspecifically bound probe molecules, specific hybridization isdetected, or even quantitated, by means of the label.

2.3 NOEY2 Polypeptide Compositions

The invention also discloses and claims a composition comprising a NOEY2tumor suppressor protein. The composition may comprises one or more hostcells which express a NOEY2 tumor suppressor protein, recombinant hostcells expresses the protein, cell suspensions, extracts, inclusionbodies, or tissue cultures or culture extracts which contain the NOEY2protein, culture supernatant, disrupted cells, cell extracts, lysates,homogenates, and the like. The compositions may be in aqueous form, oralternatively, in dry, semi-wet, or similar forms such as cell paste,cell pellets, or alternatively freeze dried, powdered, lyophilized,evaporated, or otherwise similarly prepared in dry form. Such means forpreparing tumor suppressor proteins are well-known to those of skill inthe art of protein isolation and purification. In certain embodiments,the tumor suppressor proteins may be purified, concentrated, admixedwith other reagents, or processed to a desired final form. Preferably,the composition will comprise from about 1% to about 90% by weight ofthe tumor suppressor protein, and more preferably from about 5% to about50% by weight.

In a preferred embodiment, the tumor suppressor protein compositions ofthe invention may be prepared by a process which comprises the steps ofculturing a host cell which expresses a NOEY2 tumor suppressor proteinunder conditions effective to produce such a protein, and then obtainingthe protein from the cell. The obtaining of such a tumor suppressorprotein may further include purifying, concentrating, processing, oradmixing the protein with one or more reagents. Preferably, the NOEY2tumor suppressor protein is obtained in an amount of from between about1% to about 90% by weight, and more preferably from about 5% to about70% by weight, and even more preferably from about 10% to about 20% toabout 30%, or even to about 40% or 50% by weight.

The invention also relates to a method of preparing a tumor suppressorprotein composition. Such a method generally involves the steps ofculturing a host cell which expresses a NOEY2 tumor suppressor proteinunder conditions effective to produce the protein, and then obtainingthe protein so produced. In a preferred embodiment the cell is an NIH3T3cell, or any recombinant host cell which contains a NOEY2-encoding DNAsegment. Alternatively, the recombinant plasmid vectors of the inventionmay be used to transform other suitable bacterial or eukaryotic cells toproduce the tumor suppressor protein of the invention. Eukaryotic hostcells including NIH3T3, COS7, and CAOV3, as well as yeast cells arecontemplated to be particularly useful in the preparation of the NOEY2protein. Likewise, prokaryotic host cells including Gram-negative cellssuch as E. coli, Pseudomonas spp. and related Enterobacteraceae and thelike are all contemplated to be useful in the preparation of the tumorsuppressor proteins of the invention.

In such embodiments, it is contemplated that certain advantages will begained by positioning the coding DNA segment under the control of arecombinant, or heterologous, promoter. As used herein, a recombinant orheterologous promoter is intended to refer to a promoter that is notnormally associated with a DNA segment encoding a tumor suppressorprotein or peptide in its natural environment. Such promoters mayinclude promoters normally associated with other genes, and/or promotersisolated from any bacterial, viral, or eukaryotic cell. Preferredeukaryotic cells are animal cells, with mammalian cells, particularlyhuman cells, being most preferred. Naturally, it will be important toemploy a promoter that effectively directs the expression of the DNAsegment in the cell type, tissue, organism, animal, or recombinant hostcell chosen for expression. The use of promoter and cell typecombinations for protein expression is generally known to those of skillin the art of molecular biology, for example, see Sambrook et al., 1989.The promoters employed may be constitutive, or inducible, and can beused under the appropriate conditions to direct high level expression ofthe introduced DNA segment, such as is advantageous in the large-scaleproduction of recombinant proteins or peptides. Appropriate promotersystems contemplated for use in high-level expression include, but arenot limited to, the Pichia expression vector system (Pharmacia LKBBiotechnology).

In connection with expression embodiments to prepare recombinantproteins and peptides, it is contemplated that longer DNA segments willmost often be used, with DNA segments encoding the entire peptidesequence being most preferred. However, it will be appreciated that theuse of shorter DNA segments to direct the expression of tumor suppressorpeptides or epitopic core regions, such as may be used to generateanti-tumor suppressor protein antibodies, also falls within the scope ofthe invention. DNA segments that encode peptide antigens from about 8 toabout 50 amino acids in length, or more preferably, from about 8 toabout 30 amino acids in length, or even more preferably, from about 8 toabout 20 amino acids in length are contemplated to be particularlyuseful. Such peptide epitopes may be amino acid sequences which comprisecontiguous amino acid sequences from SEQ ID NO:2.

2.4 NOEY2 Transgenes and Transformed Host Cells Expressing NOEY2

In yet another aspect, the present invention provides methods forproducing a transgenic cell, and in particular a plant or animal cellwhich expresses a nucleic acid segment encoding the novel NOEY2 tumorsuppressor protein of the present invention. The process of producingtransgenic cells is well-known in the art. In general, the methodcomprises transforming a suitable host cell with a DNA segment whichcontains a promoter operatively linked to a coding region that encodes aNOEY2 tumor suppressor protein. Such a coding region is generallyoperatively linked to a transcription-terminating region, whereby thepromoter is capable of driving the transcription of the coding region inthe cell, and hence providing the cell the ability to produce therecombinant protein in vivo. Alternatively, in instances where it isdesirable to control, regulate, or decrease the amount of a particularrecombinant tumor suppressor protein expressed in a particulartransgenic cell, the invention also provides for the expression of tumorsuppressor protein antisense mRNA. The use of antisense mRNA as a meansof controlling or decreasing the amount of a given protein of interestin a cell is well-known in the art.

In a preferred embodiment, the invention encompasses an animal cellwhich has been transformed with a nucleic acid segment of the invention,and which expresses a gene or gene segment encoding one or more of thenovel polypeptide compositions disclosed herein. As used herein, theterm “transgenic host cell” is intended to refer to a host cell, eitherprokaryotic or eukaryotic, that has incorporated DNA sequences,including but not limited to genes which are perhaps not normallypresent, DNA sequences not normally transcribed into RNA or translatedinto a protein (“expressed”), or any other genes or DNA sequences whichone desires to introduce into the non-transformed host cell, such asgenes which may normally be present in the non-transformed cell butwhich one desires to either genetically engineer or to have alteredexpression.

It is contemplated that in some instances the genome of a transgenichost cell of the present invention will have been augmented through thestable introduction of a NOEY2 transgene, either native NOEY2, orsynthetically modified or mutated NOEY2. In some instances, more thanone transgene will be incorporated into the genome of the transformedhost cell. Such is the case when more than one tumor suppressorprotein-encoding DNA segment is incorporated into the genome of such acell. In certain situations, it may be desirable to have one, two,three, four, or even more NOEY2 tumor suppressor proteins (either nativeor recombinantly-engineered) incorporated and stably expressed in thetransformed transgenic host cell. In preferred embodiments, theintroduction of the transgene into the genome of the host cell resultsin a stable integration wherein the progeny of such cells also contain acopy of the transgene in their genome.

A preferred gene which may be introduced includes, for example, a tumorsuppressor protein-encoding a DNA sequence, and particularly one or moreof the NOEY2 or NOEY2-like tumor suppressor proteins disclosed herein.Highly preferred nucleic acid sequences are those which have the nucleicacid sequence of SEQ ID NO:1 or SEQ ID NO:5, or biologically-functionalequivalents thereof, sequences which hybridize to the sequence of SEQ IDNO:1 or SEQ ID NO:5, or sequences which encode the amino acid sequenceof SEQ ID NO:2, or sequences which encode a biologically functionalequivalent protein of SEQ ID NO:2, or any of those sequences which havebeen genetically engineered to alter, modify, change, decrease orincrease the suppressor activity or specificity of the tumor suppressorprotein in such a transformed host cell.

Means for transforming a host cell and the preparation of a transgeniccell line are well-known in the art (as exemplified in U.S. Pat. Nos.5,550,318; 5,508,468; 5,482,852; 5,384,253; 5,276,269; and 5,225,341,all specifically incorporated herein by reference), and are brieflydiscussed herein. Vectors, including plasmids, cosmids, phage,phagemids, BACs (bacterial artificial chromosomes), YACs (yeastartificial chromosomes), and DNA segments for use in transforming suchcells will, of course, generally comprise either the operons, genes, orgene-derived sequences of the present invention, either native, orsynthetically-derived, and particularly those encoding the disclosedtumor suppressor proteins. These DNA constructs can further includestructures such as promoters, enhancers, polylinkers, or even genesequences which have positively- or negatively-regulating activity uponthe particular genes of interest as desired. The DNA segment or gene mayencode either a native or modified tumor suppressor protein, which willbe expressed in the resultant recombinant cells, and/or which willimpart a desired phenotype to the transformed host cell.

2.6 Compositions and Methods for Producing NOEY2-Specific Antibodies

In particular embodiments, the inventors contemplate the use ofantibodies, either monoclonal or polyclonal which specifically bind toone or more of the NOEY2 polypeptides disclosed herein. Means forpreparing and characterizing antibodies are well known in the art (See,e.g., Harlow and Lane, 1988; incorporated herein by reference). Themethods for generating monoclonal antibodies (mAbs) generally beginalong the same lines as those for preparing polyclonal antibodies.Briefly, a polyclonal antibody is prepared by immunizing an animal withan immunogenic composition in accordance with the present invention andcollecting antisera from that immunized animal. A wide range of animalspecies can be used for the production of antisera. Typically the animalused for production of anti-antisera is a rabbit, a mouse, a rat, ahamster, a guinea pig or a goat. Because of the relatively large bloodvolume of rabbits, a rabbit is a preferred choice for production ofpolyclonal antibodies.

As is well known in the art, a given composition may vary in itsimmunogenicity. It is often necessary therefore to boost the host immunesystem, as may be achieved by coupling a peptide or polypeptideimmunogen to a carrier. Exemplary and preferred carriers are keyholelimpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albuminssuch as ovalbumin, mouse serum albumin or rabbit serum albumin can alsobe used as carriers. Means for conjugating a polypeptide to a carrierprotein are well known in the art and include glutaraldehyde,m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimide andbis-biazotized benzidine.

As is also well known in the art, the immunogenicity of a particularimmunogen composition can be enhanced by the use of non-specificstimulators of the immune response, known as adjuvants. Exemplary andpreferred adjuvants include complete Freund's adjuvant (a non-specificstimulator of the immune response containing killed Mycobacteriumtuberculosis), incomplete Freund's adjuvants and aluminum hydroxideadjuvant.

The amount of immunogen composition used in the production of polyclonalantibodies varies upon the nature of the immunogen as well as the animalused for immunization. A variety of routes can be used to administer theimmunogen (subcutaneous, intramuscular, intradermal, intravenous andintraperitoneal). The production of polyclonal antibodies may bemonitored by sampling blood of the immunized animal at various pointsfollowing immunization. A second, booster, injection may also be given.The process of boosting and titering is repeated until a suitable titeris achieved. When a desired level of immunogenicity is obtained, theimmunized animal can be bled and the serum isolated and stored, and/orthe animal can be used to generate mAbs.

mAbs may be readily prepared through use of well-known techniques, suchas those exemplified in U.S. Pat. No. 4,196,265, incorporated herein byreference. Typically, this technique involves immunizing a suitableanimal with a selected immunogen composition, e.g., a purified orpartially purified tumor suppressor protein, polypeptide or peptide. Theimmunizing composition is administered in a manner effective tostimulate antibody producing cells. Rodents such as mice and rats arepreferred animals, however, the use of rabbit, sheep frog cells is alsopossible. The use of rats may provide certain advantages (Goding, 1986,pp. 60–61), but mice are preferred, with the BALB/c mouse being mostpreferred as this is most routinely used and generally gives a higherpercentage of stable fusions.

Following immunization, somatic cells with the potential for producingantibodies, specifically B lymphocytes (B cells), are selected for usein the mAb generating protocol. These cells may be obtained frombiopsied spleens, tonsils or lymph nodes, or from a peripheral bloodsample. Spleen cells and peripheral blood cells are preferred, theformer because they are a rich source of antibody-producing cells thatare in the dividing plasmablast stage, and the latter because peripheralblood is easily accessible. Often, a panel of animals will have beenimmunized and the spleen of animal with the highest antibody titer willbe removed and the spleen lymphocytes obtained by homogenizing thespleen with a syringe. Typically, a spleen from an immunized mousecontains approximately 5×10⁷ to 2×10⁸ lymphocytes.

The antibody-producing B lymphocytes from the immunized animal are thenfused with cells of an immortal myeloma cell, generally one of the samespecies as the animal that was immunized. Myeloma cell lines suited foruse in hybridoma-producing fusion procedures preferably arenon-antibody-producing, have high fusion efficiency, and enzymedeficiencies that render then incapable of growing in certain selectivemedia which support the growth of only the desired fused cells(hybridomas).

Any one of a number of myeloma cells may be used, as are known to thoseof skill in the art (Goding, pp. 65–66, 1986; Campbell, pp. 75–83,1984). For example, where the immunized animal is a mouse, one may useP3-X63/Ag8, X63-Ag8.653, NS1/1.Ag 4 1, Sp210-Ag14, FO, NSO/U, MPC-11,MPC11-X45-GTG 1.7 and S194/5XX0 Bul; for rats, one may use R210.RCY3,Y3-Ag 1.2.3, IR983F and 4B210; and U-266, GM1500-GRG2, LICR-LON-HMy2 andUC729-6 are all useful in connection with human cell fusions.

One preferred murine myeloma cell is the NS-1 myeloma cell line (alsotermed P3-NS-1-Ag4-1), which is readily available from the NIGMS HumanGenetic Mutant Cell Repository by requesting cell line repository numberGM3573. Another mouse myeloma cell line that may be used is the8-azaguanine-resistant mouse murine myeloma SP2/0 non-producer cellline.

Methods for generating hybrids of antibody-producing spleen or lymphnode cells and myeloma cells usually comprise mixing somatic cells withmyeloma cells in a 2:1 ratio, though the ratio may vary from about 20:1to about 1:1, respectively, in the presence of an agent or agents(chemical or electrical) that promote the fusion of cell membranes.Fusion methods using Sendai virus have been described (Kohler andMilstein, 1975; 1976), and those using polyethylene glycol (PEG), suchas 37% (v/v) PEG, (Gefter et al., 1977). The use of electrically inducedfusion methods is also appropriate (Goding, 1986, pp. 71–74).

Fusion procedures usually produce viable hybrids at low frequencies,about 1×10⁻⁶ to 1×10⁻⁸. However, this does not pose a problem, as theviable, fused hybrids are differentiated from the parental, unfusedcells (particularly the unfused myeloma cells that would normallycontinue to divide indefinitely) by culturing in a selective medium. Theselective medium is generally one that contains an agent that blocks thede novo synthesis of nucleotides in the tissue culture media. Exemplaryand preferred agents are aminopterin, methotrexate, and azaserine.Aminopterin and methotrexate block de novo synthesis of both purines andpyrimidines, whereas azaserine blocks only purine synthesis. Whereaminopterin or methotrexate is used, the media is supplemented withhypoxanthine and thymidine as a source of nucleotides (HAT medium).Where azaserine is used, the media is supplemented with hypoxanthine.

The preferred selection medium is HAT. Only cells capable of operatingnucleotide salvage pathways are able to survive in HAT medium. Themyeloma cells are defective in key enzymes of the salvage pathway, e.g.,hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive.The B-cells can operate this pathway, but they have a limited life spanin culture and generally die within about two wk. Therefore, the onlycells that can survive in the selective media are those hybrids formedfrom myeloma and B-cells.

This culturing provides a population of hybridomas from which specifichybridomas are selected. Typically, selection of hybridomas is performedby culturing the cells by single-clone dilution in microtiter plates,followed by testing the individual clonal supernatants (after about twoto three wk) for the desired reactivity. The assay should be sensitive,simple and rapid, such as radioimmunoassays, enzyme immunoassays,cytotoxicity assays, plaque assays, dot immunobinding assays, and thelike.

The selected hybridomas would then be serially diluted and cloned intoindividual antibody-producing cell lines, which clones can then bepropagated indefinitely to provide mAbs. The cell lines may be exploitedfor mAb production in two basic ways. A sample of the hybridoma can beinjected (often into the peritoneal cavity) into a histocompatibleanimal of the type that was used to provide the somatic and myelomacells for the original fusion. The injected animal develops tumorssecreting the specific monoclonal antibody produced by the fused cellhybrid. The body fluids of the animal, such as serum or ascites fluid,can then be tapped to provide mAbs in high concentration. The individualcell lines could also be cultured in vitro, where the mAbs are naturallysecreted into the culture medium from which they can be readily obtainedin high concentrations. mAbs produced by either means may be furtherpurified, if desired, using filtration, centrifugation and variouschromatographic methods such as HPLC or affinity chromatography.

2.7 NOEY2 Polypeptide Screening Methods and Immunodetection Kits

The present invention also provides compositions, methods and kits forscreening samples suspected of containing a NOEY2 polypeptide or a NOEY2polynucleotide that encodes such a tumor suppressor protein.Alternatively, the invention provides compositions, methods and kits forscreening samples suspected of containing tumor suppressor proteins orgenes encoding tumor suppressor proteins which are functionallyequivalent to, or substantially homologous to, the NOEY2 tumorsuppressor protein disclosed herein. Such screening may be performed onsamples such as transformed host cells, clinical or laboratory samplessuspected of containing or producing such a polypeptide or nucleic acidsegment. A kit can contain a novel nucleic acid segment or an antibodyof the present invention. The kit can contain reagents for detecting aninteraction between a sample and a nucleic acid or an antibody of thepresent invention. The provided reagent can be radio-, fluorescently- orenzymatically-labeled. The kit can contain a known radiolabeled agentcapable of binding or interacting with a nucleic acid or antibody of thepresent invention.

The reagent of the kit can be provided as a liquid solution, attached toa solid support or as a dried powder. Preferably, when the reagent isprovided in a liquid solution, the liquid solution is an aqueoussolution. Preferably, when the reagent provided is attached to a solidsupport, the solid support can be chromatograph media, a test platehaving a plurality of wells, or a microscope slide. When the reagentprovided is a dry powder, the powder can be reconstituted by theaddition of a suitable solvent, that may be provided.

In still further embodiments, the present invention concernsimmunodetection methods and associated kits. It is proposed that thetumor suppressor proteins or peptides of the present invention may beemployed to detect antibodies having reactivity therewith, or,alternatively, antibodies prepared in accordance with the presentinvention, may be employed to detect tumor suppressor proteins or tumorsuppressor protein-related epitope-containing peptides. In general,these methods will include first obtaining a sample suspected ofcontaining such a protein, peptide or antibody, contacting the samplewith an antibody or peptide in accordance with the present invention, asthe case may be, under conditions effective to allow the formation of animmunocomplex, and then detecting the presence of the immunocomplex.

In general, the detection of immunocomplex formation is quite well knownin the art and may be achieved through the application of numerousapproaches. For example, the present invention contemplates theapplication of ELISA, RIA, immunoblot (e.g., dot blot), indirectimmunofluorescence techniques and the like. Generally, immunocomplexformation will be detected through the use of a label, such as aradiolabel or an enzyme tag (such as alkaline phosphatase, horseradishperoxidase, or the like). Of course, one may find additional advantagesthrough the use of a secondary binding ligand such as a second antibodyor a biotin/avidin ligand binding arrangement, as is known in the art.

For assaying purposes, it is proposed that virtually any samplesuspected of comprising either a tumor suppressor protein or peptide ora tumor suppressor protein-related peptide or antibody sought to bedetected, as the case may be, may be employed. It is contemplated thatsuch embodiments may have application in the titering of antigen orantibody samples, in the selection of hybridomas, and the like. Inrelated embodiments, the present invention contemplates the preparationof kits that may be employed to detect the presence of tumor suppressorproteins or related peptides and/or antibodies in a sample. Samples mayinclude cells, cell supernatants, cell suspensions, cell extracts,enzyme fractions, protein extracts, or other cell-free compositionssuspected of containing tumor suppressor proteins or peptides. Generallyspeaking, kits in accordance with the present invention will include asuitable tumor suppressor protein, peptide or an antibody directedagainst such a protein or peptide, together with an immunodetectionreagent and a means for containing the antibody or antigen and reagent.The immunodetection reagent will typically comprise a label associatedwith the antibody or antigen, or associated with a secondary bindingligand. Exemplary ligands might include a secondary antibody directedagainst the first antibody or antigen or a biotin or avidin (orstreptavidin) ligand having an associated label. Of course, as notedabove, a number of exemplary labels are known in the art and all suchlabels may be employed in connection with the present invention.

The container will generally include a vial into which the antibody,antigen or detection reagent may be placed, and preferably suitablyaliquotted. The kits of the present invention will also typicallyinclude a means for containing the antibody, antigen, and reagentcontainers in close confinement for commercial sale. Such containers mayinclude injection or blow-molded plastic containers into which thedesired vials are retained.

2.8 NOEY2-Derived Epitopic Sequences

The present invention is also directed to NOEY2 protein or peptidecompositions, free from total cells and other peptides, which comprise apurified NOEY2 protein or peptide which incorporates an epitope that isimmunologically cross-reactive with one or more anti-tumor suppressorprotein antibodies. In particular, the invention concerns epitopic coresequences derived from NOEY2 and NOEY2-derived proteins or peptides.

As used herein, the term “incorporating an epitope(s) that isimmunologically cross-reactive with one or more anti-tumor suppressorprotein antibodies” is intended to refer to a peptide or protein antigenwhich includes a primary, secondary or tertiary structure similar to anepitope located within a tumor suppressor protein or polypeptide. Thelevel of similarity will generally be to such a degree that monoclonalor polyclonal antibodies directed against the tumor suppressor proteinor polypeptide will also bind to, react with, or otherwise recognize,the cross-reactive peptide or protein antigen. Various immunoassaymethods may be employed in conjunction with such antibodies, such as,for example, Western blotting, ELISA, RIA, and the like, all of whichare known to those of skill in the art.

The identification of NOEY2 immunodominant epitopes, and/or theirfunctional equivalents, suitable for use in vaccines is a relativelystraightforward matter. For example, one may employ the methods of Hopp,as taught in U.S. Pat. No. 4,554,101, incorporated herein by reference,which teaches the identification and preparation of epitopes from aminoacid sequences on the basis of hydrophilicity. The methods described inseveral other papers, and software programs based thereon, can also beused to identify epitopic core sequences (see, e.g., Jameson and Wolf,1988; Wolf et al., 1988; U.S. Pat. No. 4,554,101). The amino acidsequence of these “epitopic core sequences” may then be readilyincorporated into peptides, either through the application of peptidesynthesis or recombinant technology.

Preferred peptides for use in accordance with the present invention willgenerally be on the order of about 8 to about 20 amino acids in length,and more preferably about 8 to about 15 amino acids in length. It isproposed that shorter antigenic tumor suppressor protein-derivedpeptides will provide advantages in certain circumstances, for example,in the preparation of immunologic detection assays. Exemplary advantagesinclude the ease of preparation and purification, the relatively lowcost and improved reproducibility of production, and advantageousbiodistribution.

It is proposed that particular advantages of the present invention maybe realized through the preparation of synthetic peptides which includemodified and/or extended epitopic/immunogenic core sequences whichresult in a “universal” epitopic peptide directed to tumor suppressorproteins, and in particular NOEY2 and NOEY2-related sequences. Theseepitopic core sequences are identified herein in particular aspects ashydrophilic regions of the particular polypeptide antigen. It isproposed that these regions represent those which are most likely topromote T-cell or B-cell stimulation, and, hence, elicit specificantibody production.

An epitopic core sequence, as used herein, is a relatively short stretchof amino acids that is “complementary” to, and therefore will bind,antigen binding sites on the tumor suppressor protein-directedantibodies disclosed herein. Additionally or alternatively, an epitopiccore sequence is one that will elicit antibodies that are cross-reactivewith antibodies directed against the peptide compositions of the presentinvention. It will be understood that in the context of the presentdisclosure, the term “complementary” refers to amino acids or peptidesthat exhibit an attractive force towards each other. Thus, certainepitope core sequences of the present invention may be operationallydefined in terms of their ability to compete with or perhaps displacethe binding of the desired protein antigen with the correspondingprotein-directed antisera.

In general, the size of the polypeptide antigen is not believed to beparticularly crucial, so long as it is at least large enough to carrythe identified core sequence or sequences. The smallest useful coresequence anticipated by the present disclosure would generally be on theorder of about 8 amino acids in length, with sequences on the order of10 to 20 being more preferred. Thus, this size will generally correspondto the smallest peptide antigens prepared in accordance with theinvention. However, the size of the antigen may be larger where desired,so long as it contains a basic epitopic core sequence.

The identification of epitopic core sequences is known to those of skillin the art, for example, as described in U.S. Pat. No. 4,554,101,incorporated herein by reference, which teaches the identification andpreparation of epitopes from amino acid sequences on the basis ofhydrophilicity. Moreover, numerous computer programs are available foruse in predicting antigenic portions of proteins (see e.g., Jameson andWolf, 1988; Wolf et al., 1988). Computerized peptide sequence analysisprograms (e.g., DNAStar® software, DNAStar, Inc., Madison, Wis.) mayalso be useful in designing synthetic peptides in accordance with thepresent disclosure.

Syntheses of epitopic sequences, or peptides which include an antigenicepitope within their sequence, are readily achieved using conventionalsynthetic techniques such as the solid phase method (e.g., through theuse of commercially available peptide synthesizer such as an AppliedBiosystems Model 430A Peptide Synthesizer). Peptide antigens synthesizedin this manner may then be aliquotted in predetermined amounts andstored in conventional manners, such as in aqueous solutions or, evenmore preferably, in a powder or lyophilized state pending use.

In general, due to the relative stability of peptides, they may bereadily stored in aqueous solutions for fairly long periods of time ifdesired, e.g., up to six months or more, in virtually any aqueoussolution without appreciable degradation or loss of antigenic activity.However, where extended aqueous storage is contemplated it willgenerally be desirable to include agents including buffers such as Trisor phosphate buffers to maintain a pH of about 7.0 to about 7.5.Moreover, it may be desirable to include agents which will inhibitmicrobial growth, such as sodium azide or Merthiolate. For extendedstorage in an aqueous state it will be desirable to store the solutionsat about 4° C., or more preferably, frozen. Of course, where thepeptides are stored in a lyophilized or powdered state, they may bestored virtually indefinitely, e.g., in metered aliquots that may berehydrated with a predetermined amount of water (preferably distilled)or buffer prior to use.

3.0 BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1A. Complete NOEY2 cDNA nucleotide sequence (SEQ ID NO:1) and thededuced amino acid sequence (SEQ ID NO:2) of the NOEY2 protein. Anasterisk indicates the stop codon.

FIG. 1B. Continuation of sequence of Complete NOEY2 cDNA nucleotidesequence (SEQ ID NO:1) and the deduced amino acid sequence (SEQ ID NO:2)of the NOEY2 protein.

FIG. 2. The pairwise amino acid sequence comparisons of NOEY2 with Rasand Rap family members. Four GDP/GTP binding domains and the CAAX motifare indicated by underlining. The bold type indicates residues conservedin nearly all GTPases.

FIG. 3A. Northern blot analysis of NOEY2 in cells and tissues. NOEY2cDNA probe was labeled with ³²P-dCTP by random primer. Fifteenmicrograms of total cellular RNA was separated in 1.2%formaldehyde-agarose gels and immobilized on a Hybond-N⁺ membrane(Amersham) by standard capillary transfer and UV crosslinking, and thenprehybridized and hybridized to NOEY2 probe in 50% formamide, 1×SSC, 10×Denhardt's solution, 10 mM EDTA, 0.1% SDS and 300 μg/ml denatured salmonsperm DNA at 42° C. for 24 h. Hybridization of the same blot to a probefor 18S-rRNA indicates an equal amount of RNA in all lanes. OSE cells(lane 1 to 3) and ovarian cancer cell lines (lane 4 to 9). The primarynormal OSE cells culture were obtained by gently scraping the surface ofthe ovaries which were from the patients undergoing surgery fornonmalignant gynecological diseases. The scraped epithelial cells werecultured in OSE medium (MCDB105/199 medium supplemented with 15% fetalcalf serum and 10 ng/ml Epidermal Growth Factor). OSE cells were stainedby antibody against cytokeratins. Ovarian cancer cell lines culture wasas described before (Kruk et al., 1990).

FIG. 3B. Normal breast epithelial cells (NBE) cancer cell lines (lanes 1to 4) and breast cancer cell lines (lanes 5 to 12).

FIG. 3C. OSE cells (lane 1 to 3) and ovarian cancer patients' ascitescells (lane 4 to 9). Ovarian cancer cells primary cultures were obtainedby separating tumor cells from ovarian cancer patients' ascites.Patients' ascites cells were thawed, centrifuged and resuspended in 2 mlstock Iso-osmotic Percoll (SIP) and placed in 15 ml tubes, then usingPasteur pipettes, 2 ml each of five different diluted SIP (the densitieswere 1.070, 1.058, 1.047, 1.035 and 1.023) were carefully layered toform a gradient. Gradients were centrifuged at 1500 rpm for 20 min usinga swinging bucket centrifuge. Most ascites samples contain tumor cellsthat fraction near the second and third interfaces of the percollgradient. H & E stains were performed to check the composition of thefractions. After processing ascites by percoll density gradient, furtherpurification was obtained using magnetic beads coated with CD45. Thepurified cancer cells were stained by five monoclonal antibodies whichspecifically reacted with ovarian cancer antigens.

FIG. 3D. Multiple human tissues (Clonetech). 2 μg poly-RNA each line.Hybridization was done according to the methods of the manufacturer.

FIG. 3E. Western blot of 26-kDa protein, NIH3T3-NOEY2 (positive control(lane 1), OSE (lanes 2 to 4), ovarian cancer cell lines (lanes 5 to 9).

FIG. 4A. NOEY2-induced growth inhibition of ovarian and breast cancercell lines. Colony formation after NOEY2 cDNA transfection. (a. CarrierDNA only; b. pcDNA3 vector only; c. pcDNA3 vector with NOEY2 in theantisense orientation; d. pcDNA3 vector with NOEY2 in the senseorientation).

FIG. 4B. NOEY2-induced growth inhibition of ovarian and breast cancercell lines. Inhibition of cyclin D1 promoter activity in Saos-2, NIH3T3,SKBr3 and Hey cells. NOEY2 sense and antisense constructs wereco-transfected with a luciferase reporter under the control of the humancyclin D1 promoter (Albanese et al., 1995). Luciferase activity measuredin cells transfected with the sense construct was expressed as apercentage of activity measured in cells transfected with the antisense.The results were from representative studies performed in triplicate.

FIG. 5A. NBE 007 cell proliferation as measured by MTT (Ferrari et al.,1990). Primary cultures of NBE 007 were grown under two conditions: OSEmedium or MEGM medium (Clonetics) which contains EGF, insulin,hydrocortisone and BPE.

FIG. 5B. Expression of NOEY2 and p21^(WAF1/CIP1) in OSE and NBE culturesas assessed by Northern analysis.

FIG. 6A. Induction ofp₂₁ ^(WAF1/CIP1) expression by HA-NOEY2.

FIG. 6B. Induction ofp₂₁ ^(WAF1/CIP1) expression by HA-NOEY2. HA-NOEY2transfectants.

FIG. 6C. HA-Erk2 transfectants. P21^(WAF1/CIP1) indicated in green(FITC).

FIG. 6D. HA indicated in red (rhodamine).

FIG. 7. Map of the genomic structure of NOEY2. Exons 1 and 2 are show aswell as the large intron 1. Nucleotide residue numbers are shown below.

FIG. 8. Expression in OSE.NBE and ovarian cancer cell lines (SKOv3 andHey) of the wild type NOEY2 promoter linked to a luciferase reporter(p9) and the NOEY2 promoter with an A to G mutation at −750 (p9m 2-1.1and p9m 4-1. An A to G mutation was introduced into the wild type NOEY2promoter by site directed mutagenesis. The results are fromrepresentative studies performed in triplicate.

4.0 DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

4.1 Molecular Alterations in Epithelial Ovarian Cancer

Studies from the inventors' group (Jacobs et al., 1992) and others (Moket al., 1992; Li et al., 1993) have demonstrated that more than 90% ofepithelial ovarian cancers are clonal based on the similarity ofX-chromosome inactivation, p53 mutation and loss of heterozygosity inthe primary tumor and different metastases. If this is the case,multiple mutations must occur within a single epithelial cell to producemalignant transformation. Mutations can be carried in the germ line oroccur somatically. Germline mutation of several genes has recently beenimplicated in familial ovarian cancer including BRCA1 (Miki et al.,1994), BRCA2 (Wooster et al., 1994) mismatch repair genes (Lynch et al.,1996) and, on rare occasions, p53 (Malkin et al., 1990). For more than adecade the inventors' laboratory has focused on defining the somaticchanges that distinguish malignant from benign ovarian epithelium (Bastet al., 1992). With Larry Feig and Geoff Cooper, the inventors had founda mutant Ki-Ras gene in ovarian cancer cells, but not in benignmesothelial cells from the same patient's ascites (Feig et al., 1984),demonstrating somatic mutation of Ras in a human tumor. Subsequentstudies have found mutated or amplified Ki-, Ha- or N-Ras in less than15% of ovarian cancers, most notably in mucinous or borderline lesions.Interestingly, as described below, Ras is constitutively activated in asignificant fraction of ovarian cancer cell lines in the absence ofmutation or amplification. In such cancers, signaling can be documentedthrough the MAP kinase pathway and the ets-2 transcription factor,possibly accounting for the larger fraction of ovarian cancers thatoverexpress c-myc and other Ras-responsive genes such as urokinaseplasminogen activator and matrix metalloproteinases that are importantfor invasion.

Physiologic activation of Ras implies that receptors and/or transducingmolecules upstream are activated. A number of autocrine and paracrinegrowth regulatory pathways have been identified that appear to bealtered in epithelial ovarian cancers when compared to normal ovarianepithelium. The EGFR family (EGFR, HER-2, HER-3, HER-4) of tyrosinekinase growth factor receptors may be particularly important in ovariancancer. Normal ovarian surface epithelium expresses EGFR and can bestimulated to proliferate with exogenous EGF or TGF-(Rodriguez et al.,1991). Ovarian carcinomas retain EGFR in 70% of cases (Berchuck et al.,1991) and can secrete EGF and TGF-. Evidence for autocrine growthregulation has been obtained by inhibiting proliferation of ovariancancer cell lines with anti-TGF-antibodies both in vitro and in vivo(Stromberg et al., 1992; Morishige et al., 1991). The inventors havedemonstrated that loss of EGFR expression by tumor cells is associatedwith a slight, but statistically significant improvement in survival(Berchuck et al., 1991), consistent with loss of autocrine growthstimulation. Overexpression of HER-2 (c-erbB2) has been observed in 30%of cases of ovarian cancer. In stage III disease, overexpression ofHER-2 is associated with a significantly shortened survival (Berchuck etal., 1990), although the significance of this marker in early stagedisease is less certain (Rubin et al., 1993; Kacinski et al., 1992).Expression of HER-3 is increased in some borderline and early invasiveovarian tumors. Little has been reported regarding HER-4 in clinicalmaterial. Studies in cell culture suggest that the ligands heregulin andneu differentiating factor (NDF) signal through homodimers of HER-4 orheterodimers containing of HER-2/HER-3 or HER-2/HER-4. Cross-talkbetween the EGFR family members has been described. The inventors' ownstudies suggest that the ratio of HER-2/HER-3 may be particularlyimportant in determining whether growth of tumor cells is stimulated orinhibited by heregulin. Counterintuitively, heregulin and NDF caninhibit, rather than stimulate clonogenic growth of ovarian and breastcancer cells that overexpress HER-2 in the presence of modest levels ofHER-3 or HER-4 (Xu et al., 1996). Although tumor cells that overexpressHER-2 are growth inhibited by these ligands or by agonistic antibodiesthat bind only to HER-2, both ligands and antibodies can stimulate theability of ovarian or breast cancer cells to invade matrigel membranes,associated with increased expression of matrix metalloproteinase 9(MMP9) (Xu et al., 1994). Consequently, overexpression of HER-2 maypotentiate the ability of tumor cells to invade and metastasize ratherthan to proliferate.

Other tyrosine kinase growth factor receptors have been identified inovarian cancers that can signal through Ras and other pathways. Normalovarian surface epithelium expresses little, if any of the fms tyrosinekinase growth factor receptor and secretes low levels of its ligandmacrophage colony stimulating factor (M-CSF or CSF-1) (Lidor et al.,1993). Approximately 50% of ovarian cancers express fms (Kacinski, 1995)and 70% secrete substantial levels of M-CSF (Xu et al., 1991),consistent with possible autocrine growth stimulation through the Raspathway. In addition, M-CSF is a potent chemoattractant for macrophages(Dorsch et al., 1993) which release a number of cytokines with growthregulatory activity for normal and transformed ovarian epitheliumincluding tumor necrosis factor alpha (TNF-α), IL-1 and IL-6 (Wu et al.,1992). In the context of this P01, Dr. Mills (Project 4) is studyingOCAF, a novel lysophospholipid growth factor that is present in ascitesfluid from a majority of ovarian cancer patients and stimulates tumorgrowth in more than 90% of cases. OCAF activates the MAP kinase pathwaythrough a cascade involving Ras and tyrosine kinases. Dr. Skinner(Project 1) is evaluating the role of several peptide factors includingKGF, HGF and kit ligand that activate receptors which can also impingeon the Ras pathway.

Growth inhibition of ovarian surface epithelium is mediated byTGF-(Berchuck et al., 1992) and other factors elaborated by theunderlying stroma (Karlan et al., 1995). The inventors' group has shownthat TGF-1 and 2 can be expressed by and activated in normal ovarianepithelial cells consistent with autocrine as well as paracrine growthinhibition (Berchuck et al., 1992). Different ovarian cancer cell lineshave lost the ability to express, activate or respond to TGF-(Berchucket al., 1992), but more than 90% ovarian cancer ascites tumor specimenscan inhibited by TGF-and a fraction undergo apoptosis (Hurteau et al.,1994). By contrast, normal epithelial cells can be growth inhibited butdo not undergo apoptosis in response to TGF-(Havrilesky et al., 1995).Consequently, TGF- may provide a primitive surveillance mechanism foreliminating epithelial cells as they transform. Expression of TGF- islost in up to 40% of ovarian cancer specimens that presumably would havelost autocrine growth inhibition, but paracrine growth inhibition andinduction of apoptosis could be obtained from TGF-secreted by theunderlying stroma.

Other candidates for negative growth regulation of ovarian epithelialcells include the protein tyrosine phosphatases (PTPs) that candeactivate or reverse the effects of certain tyrosine kinases. PTPs can,however, stimulate as well as inhibit growth of cells in differentlineages. The inventors' group has cloned fragments of 13 PTPs fromovarian cancers (Wiener et al., 1996). PTP-1C and PTP-2A were regularlyexpressed in normal epithelial cells, but not expressed in a fraction oftumors, whereas PTP-1B, PTP- and PTP-H are upregulated in response totransfection of HER-2 (Wiener et al., 1996). Evidence is mounting forthe potential role of PTP-1C as a tumor suppressor in several differentcell lineages (Shultz et al, 1993) and the inventors' own data indicatethat expression of PTP-1C inhibits growth of ovarian cancer cells. Inclinical material a correlation has been observed between expression ofPTP-1B and that of EGFR, HER-2 and fms (Wiener et al., 1994). Inexperimental systems PTP-1B can suppress transformation induced byexpression of mutant HER-2 (Brown-Shiner et al., 1992). Overexpressionof PTP-1B may reflect an inadequate homeostatic mechanism in ovariancarcinomas that have persistent or increased expression of tyrosinekinases such as EGFR, HER-2 and fms. Alternatively, the PTP-1B promotermay contain response elements for signaling pathways activated bytyrosine kinase receptors.

4.2 Tumor Suppressor Genes in Epithelial Ovarian Cancer

As in cancers that arise at other sites, the tumor suppressor gene beststudied in ovarian cancer is p53 which is mutated in approximately 50%of metastatic tumors, but in only 15% of lesions in stage IA or IB(Berchuck et al., 1994). The gene is rarely affected in benign orborderline lesions. As the inventors had shown with Drs. Matt Kohler andAndrew Berchuck, the pattern of p53 mutation is most consistent withspontaneous deamination during normal replication rather than formationof adducts with exogenous carcinogens (Kohler et al., 1993). Loss ofheterozygosity at RB has been observed in a fraction of ovarian cancers,but functional RB protein is generally present.

Aside from p53 and RB, little is known about the role of many of thepreviously identified tumor suppressor genes in ovarian cancer.Moreover, no suppressor gene specific to ovarian cancer has beenisolated. Although a number of approaches have been used to identifyputative tumor suppressor genes, the inventors have utilizeddifferential display of mRNA by means of the polymerase chain reaction(DDPCR™) to isolate NOEY2, a novel Ras-related gene, that may serve as atumor suppressor gene in ovarian cancer. A substantial literature hasaddressed the possibility that Ras-related genes might inhibit Rasfunction directly or indirectly.

4.3 RAS and RAP Families

A large superfamily (>50 members) of monomeric GTP-binding proteinsstructurally related to the Ras oncogene proteins has been described inthe past few years. The model of action of Ras has recently beenintensively investigated, and one of its direct downstream targetmolecules has been identified to be c-raf-1, which induces theactivation of the MAP kinase/ERK through MEK. The Rap family consists ofseveral highly homologous members—Rap1A, Rap1B, and Rap2—that belong tothe Ras superfamily of small GTP-binding proteins (Pizon et al., 1988).Rap1A and/or Rap1B have been shown to antagonize the Ras Functions, suchas the Ki-Ras-induced transformation of NIH 3T3 cells (Kitayama et al.,1989), the Ha-Ras-induced germinal vesicle breakdown in Xenopus oocytes(Campa et al., 1991), the N-Ras-inhibited muscarinic K⁺ channel activity(Yatani et al., 1991), the Ki-Ras-induced activation of the c-fospromoter/enhancer in NIH 3T3 cells (Sakoda et al., 1992), theproliferation of middle T antigen-transformed Rat-2 cells (Jelinek andHassell, 1992), and the Ha-Ras-induced activation of the c-Raf-1 proteinkinase-dependent Map kinase cascade in Rat-1 cells (Cook et al., 1993).Rap1A and/or Rap1B have been shown to be phosphorylated by proteinkinase A in both intact cells and cell-free systems (Quilliam et al.,1991), by Ca²⁺/calmodulin-dependent protein kinase Gr in a cell-freesystem (Sahyoun et al., 1991), and by protein kinase G in a cell-freesystem (Miura et al., 1992). The protein kinase A-catalyzedphosphorylation sites of Rap1A and Rap1B are Ser 180 and Ser 179,respectively, in their C-terminal regions (Quilliam et al., 1991; Hataet al., 1991). This phosphorylation of Rap1B lowers its membrane bindingactivity and induces its translocation from the membrane to the cytosol(Hata et al., 1991). The phosphorylation of Rap1B makes it sensitive tothe action of 5 mg GDS to stimulate its GDP/GTP exchange reaction (Hataet al., 1991). These observations suggest that Rap1 has multiplefunctions, but the mode of action of Rap1 at a molecular level remainsto be clarified. Of particular interest is the possibility that Rapfamily members may not only antagonize Ras, but also signal independentof the Ras pathway.

The GTP binding site of Ras proteins consists of four non-contiguousregions encountered in all the proteins of the Ras superfamily. Amongthese regions six amino acids: DTAGQE, in positions 57 to 62 of theK-Ras protein, seemed to be a hallmark of all the Ras and Ras-relatedproteins. It is known that single amino acid substitution in p21Ras ataa12 (glycine), 13 (glycine) and 61 (glutamine) significantly reduce theintrinsic GTPase activity of Ras proteins and prevent Ras-GAP fromaccelerating the rate of GTP hydrolysis. Therefore, it appears that thisdomain plays an essential role in the control of the biologicalproperties of the Ras proteins. Random mutagenesis studies also shownthat amino acid substitutions at positions 59 and 63 can activate Rastransforming potential.

4.4 Loss of Heterozygosity in Ovarian Cancers

Based upon the paradigm of the RB gene, LOH has been observed withincancers at the site of deleted tumor suppressor genes. Numerous studiesof loss of heterozygosity (LOH) in ovarian carcinoma have been publishedsince 1989 and are all largely based on techniques using normal-tumorpairs. Sites of LOH have been reported on 1p, 3p, 4p, 6p, 7p, 8q, 11p,13q, 17p, 17q, 18q and 22. Studies have detected LOH at sites distal toBRCA1 on chromosome 17q (Hacibs et al., 1993) and at the BRCA2 site onchromosome 13q. The short arm of chromosome 1 is frequently affected byrearrangements in a variety of human malignancies. Genetic alterations,predominantly deletions, which are indicative of the presence of aputative tumor suppressor gene at chromosome 1p, are observed in ovarianand breast cancer. Dr. Gray (Project 3) has detected LOH at 1p31 inovarian cancer, as has Drs. John Lancaster and Andrew Berchuck. Inbreast cancer LOH is found at 1p31 (Stromberg et al., 1992) in 28% to50% of cases (Nagai et al., 1995; Loupart et al., 1995; Hoggard et al.,1995). In addition to NOEY2, several other genes map to chromosome1p31-32 that could suppress growth, including VCAM-1 which functions asan adhesion molecule and p18 which inhibits the cyclin D-CDK6 complex.

4.5 Protein Isolation and Purification

In certain embodiments it may be desirable to purify NOEY2 polypeptides,NOEY2 epitopes, NOEY2-derived peptide fragments, or variants thereof.Protein purification techniques are well known to those of skill in theart. These techniques involve, at one level, the crude fractionation ofthe cellular milieu to polypeptide and non-polypeptide fractions. Havingseparated the polypeptide from other proteins, the polypeptide ofinterest may be further purified using chromatographic andelectrophoretic techniques to achieve partial or complete purification(or purification to homogeneity). Analytical methods particularly suitedto the preparation of a pure peptide are ion-exchange chromatography,exclusion chromatography; polyacrylamide gel electrophoresis;isoelectric focusing. A particularly efficient method of purifyingpeptides is fast protein liquid chromatography or even HPLC.

Certain aspects of the present invention concern the purification, andin particular embodiments, the substantial purification, of an encodedprotein or peptide. The term “purified protein or peptide” as usedherein, is intended to refer to a composition, isolatable from othercomponents, wherein the protein or peptide is purified to any degreerelative to its naturally-obtainable state. A purified protein orpeptide therefore also refers to a protein or peptide, free from theenvironment in which it may naturally occur.

Generally, “purified” will refer to a protein or peptide compositionthat has been subjected to fractionation to remove various othercomponents, and which composition substantially retains its expressedbiological activity. Where the term “substantially purified” is used,this designation will refer to a composition in which the protein orpeptide forms the major component of the composition, such asconstituting about 50%, about 60%, about 70%, about 80%, about 90%,about 95% or more of the proteins in the composition.

Various methods for quantifying the degree of purification of theprotein or peptide will be known to those of skill in the art in lightof the present disclosure. These include, for example, determining thespecific activity of an active fraction, or assessing the amount ofpolypeptides within a fraction by SDS/PAGE analysis. A preferred methodfor assessing the purity of a fraction is to calculate the specificactivity of the fraction, to compare it to the specific activity of theinitial extract, and to thus calculate the degree of purity, hereinassessed by a “-fold purification number.” The actual units used torepresent the amount of activity will, of course, be dependent upon theparticular assay technique chosen to follow the purification and whetheror not the expressed protein or peptide exhibits a detectable activity.

Various techniques suitable for use in protein purification will be wellknown to those of skill in the art. These include, for example,precipitation with ammonium sulfate, PEG, antibodies and the like or byheat denaturation, followed by centrifugation; chromatography steps suchas ion exchange, gel filtration, reverse phase, hydroxylapatite andaffinity chromatography; isoelectric focusing; gel electrophoresis; andcombinations of such and other techniques. As is generally known in theart, it is believed that the order of conducting the variouspurification steps may be changed, or that certain steps may be omitted,and still result in a suitable method for the preparation of asubstantially purified protein or peptide.

There is no general requirement that the protein or peptide always beprovided in their most purified state. Indeed, it is contemplated thatless substantially purified products will have utility in certainembodiments. Partial purification may be accomplished by using fewerpurification steps in combination, or by utilizing different forms ofthe same general purification scheme. For example, it is appreciatedthat a cation-exchange column chromatography performed utilizing an HPLCapparatus will generally result in a greater “-fold” purification thanthe same technique utilizing a low pressure chromatography system.Methods exhibiting a lower degree of relative purification may haveadvantages in total recovery of protein product, or in maintaining theactivity of an expressed protein.

It is known that the migration of a polypeptide can vary, sometimessignificantly, with different conditions of SDS/PAGE (Capaldi et al.,1973; Capaldi et al., 1974; Capaldi et al., 1977). It will therefore beappreciated that under differing electrophoresis conditions, theapparent molecular weights of purified or partially purified expressionproducts may vary.

High performance liquid chromatography (HPLC) is characterized by a veryrapid separation with extraordinary resolution of peaks. This isachieved by the use of very fine particles and high pressure to maintainan adequate flow rate. Separation can be accomplished in a matter ofmin, or at most an h. Moreover, only a very small volume of the sampleis needed because the particles are so small and close-packed that thevoid volume is a very small fraction of the bed volume. Also, theconcentration of the sample need not be very great because the bands areso narrow that there is very little dilution of the sample.

Gel chromatography, or molecular sieve chromatography, is a special typeof partition chromatography that is based on molecular size. The theorybehind gel chromatography is that the column, which is prepared withtiny particles of an inert substance that contain small pores, separateslarger molecules from smaller molecules as they pass through or aroundthe pores, depending on their size. As long as the material of which theparticles are made does not adsorb the molecules, the sole factordetermining rate of flow is the size. Hence, molecules are eluted fromthe column in decreasing size, so long as the shape is relativelyconstant. Gel chromatography is unsurpassed for separating molecules ofdifferent size because separation is independent of all other factorssuch as pH, ionic strength, temperature, etc. There also is virtually noadsorption, less zone spreading and the elution volume is related in asimple matter to molecular weight.

Affinity chromatography is a chromatographic procedure that relies onthe specific affinity between a substance to be isolated and a moleculethat it can specifically bind to. This is a receptor-ligand typeinteraction. The column material is synthesized by covalently couplingone of the binding partners to an insoluble matrix. The column materialis then able to specifically adsorb the substance from the solution.Elution occurs by changing the conditions to those in which binding willnot occur (alter pH, ionic strength, temperature, etc.).

A particular type of affinity chromatography useful in the purificationof carbohydrate containing compounds is lectin affinity chromatography.Lectins are a class of substances that bind to a variety ofpolysaccharides and glycoproteins. Lectins are usually coupled toagarose by cyanogen bromide. Conconavalin A coupled to Sepharose was thefirst material of this sort to be used and has been widely used in theisolation of polysaccharides and glycoproteins other lectins that havebeen include lentil lectin, wheat germ agglutinin which has been usefulin the purification of N-acetyl glucosaminyl residues and Helix pomatialectin. Lectins themselves are purified using affinity chromatographywith carbohydrate ligands. Lactose has been used to purify lectins fromcastor bean and peanuts; maltose has been useful in extracting lectinsfrom lentils and jack bean; N-acetyl-D-galactosamine is used forpurifying lectins from soybean; N-acetylglucosamine binds to lectinsfrom wheat germ; D-galactosamine has been used in obtaining lectins fromclams and L-fucose will bind to lectins from lotus.

The matrix should be a substance that itself does not adsorb moleculesto any significant extent and that has a broad range of chemical,physical and thermal stability. The ligand should be coupled in such away as to not affect its binding properties. The ligand should alsoprovide relatively tight binding. And it should be possible to elute thesubstance without destroying the sample or the ligand. One of the mostcommon forms of affinity chromatography is immunoaffinitychromatography. The generation of antibodies that would be suitable foruse in accord with the present invention is discussed below.

4.6 Synthetic NOEY2 and NOEY2-Derived Peptides

To achieve certain objectives of the invention, it is desirable toprepare NOEY2-derived peptides and polypeptide fragments for use invarious diagnostic and therapeutic applications. Because of theirrelatively small size, the peptides of the invention can also besynthesized in solution or on a solid support in accordance withconventional techniques. Various automatic synthesizers are commerciallyavailable and can be used in accordance with known protocols. See, forexample, Stewart and Young, (1966); Voss et al., (1983); Merrifield,(1986); and Barany and Merrifield (1979), each incorporated herein byreference. Short peptide sequences, or libraries of overlappingpeptides, usually from about 6 or so amino acids, and up to andincluding about 35 to 50 or so amino acids, which correspond to theselected regions described herein, can be readily synthesized and thenscreened in screening assays designed to identify reactive peptides.Alternatively, recombinant DNA technology may be employed wherein anucleotide sequence which encodes a peptide of the invention is insertedinto an expression vector, transformed or transfected into anappropriate host cell and cultivated under conditions suitable forexpression.

4.7 NOEY2-Derived Antigen Compositions

The present invention also provides for the use of NOEY2 proteins orpeptides as antigens for the immunization of animals relating to theproduction of antibodies. It is envisioned that either NOEY2, orportions thereof, will be coupled, bonded, bound, conjugated orchemically-linked to one or more agents via linkers, polylinkers orderivatized amino acids. This may be performed such that a bispecific ormultivalent composition or vaccine is produced. It is further envisionedthat the methods used in the preparation of these compositions will befamiliar to those of skill in the art and should be suitable foradministration to animals, i.e., pharmaceutically acceptable. Preferredagents are the carriers are keyhole limpet hemocyannin (KLH) or bovineserum albumin (BSA).

4.8 Antisense Constructs

In some cases, mutant tumor suppressors may not be non-functional.Rather, they may have aberrant functions that cannot be overcome byreplacement gene therapy, even where the “wild-type” molecule isexpressed in amounts in excess of the mutant polypeptide. Therefore, animportant aspect of the invention concerns the preparation and use ofNOEY2 antisense constructs. Such antisense technology may be used to“knock-out” or reduce the function or expression of NOEY2 in a cell, ormay ablate the function of NOEY2 in the development of cell line or in atransgenic mouse or other animal used in research, or diagnostic and/orscreening methods.

The methodology for antisense techniques is well-known to molecularbiologists. In a general sense, antisense methods take advantage of thefact that nucleic acids tend to pair with “complementary” sequences. Bycomplementary, it is meant that polynucleotides are those which arecapable of base-pairing according to the standard Watson-Crickcomplementarity rules. That is, the larger purines will base pair withthe smaller pyrimidines to form combinations of guanine paired withcytosine (G:C) and adenine paired with either thymine (A:T) in the caseof DNA, or adenine paired with uracil (A:U) in the case of RNA.Inclusion of less common bases such as inosine, 5-methylcytosine,6-methyladenine, hypoxanthine and others in hybridizing sequences doesnot interfere with pairing.

Targeting double-stranded (ds) DNA with polynucleotides leads totriple-helix formation; targeting RNA will lead to double-helixformation. Antisense polynucleotides, when introduced into a targetcell, specifically bind to their target polynucleotide and interferewith transcription, RNA processing, transport, translation and/orstability. Antisense RNA constructs, or DNA encoding such antisenseRNA's, may be employed to inhibit gene transcription or translation orboth within a host cell, either in vitro or in vivo, such as within ahost animal, including a human subject.

Antisense constructs may be designed to bind to the promoter and othercontrol regions, exons, introns or even exon-intron boundaries of agene. It is contemplated that the most effective antisense constructswill include regions complementary to intron/exon splice junctions.Thus, it is proposed that a preferred embodiment includes an antisenseconstruct with complementarity to regions within 50–200 bases of anintron-exon splice junction. It has been observed that some exonsequences can be included in the construct without seriously affectingthe target selectivity thereof. The amount of exonic material includedwill vary depending on the particular exon and intron sequences used.One can readily test whether too much exon DNA is included simply bytesting the constructs in vitro to determine whether normal cellularfunction is affected or whether the expression of related genes havingcomplementary sequences is affected.

As stated above, “complementary” or “antisense” means polynucleotidesequences that are substantially complementary over their entire lengthand have very few base mismatches. For example, sequences of fifteenbases in length may be termed complementary when they have complementarynucleotides at thirteen or fourteen positions. Naturally, sequenceswhich are completely complementary will be sequences which are entirelycomplementary throughout their entire length and have no basemismatches. Other sequences with lower degrees of homology also arecontemplated. For example, an antisense construct which has limitedregions of high homology as well as non-homologous regions (e.g.,ribozyme) could be designed. These molecules, though having less than50% homology, would bind to target sequences under appropriateconditions. The preparation and use of such ribozymes are described indetail in the following section.

In some circumstances, it may be advantageous to combine portions ofgenomic DNA with cDNA or synthetic sequences to generate specificconstructs. For example, where an intron is desired in the ultimateconstruct, a genomic clone will need to be used. The cDNA or asynthesized polynucleotide may provide more convenient restriction sitesfor the remaining portion of the construct and, therefore, would be usedfor the rest of the sequence.

4.9 Ribozymes

Another approach for addressing the “dominant negative” mutant tumorsuppressor is through the use of ribozymes. Although proteinstraditionally have been used for catalysis of nucleic acids, anotherclass of macromolecules has emerged as useful in this endeavor.Ribozymes are RNA-protein complexes that cleave nucleic acids in asite-specific fashion. Ribozymes have specific catalytic domains thatpossess endonuclease activity (Kim and Cech, 1987; Gerlach et al., 1987;Forster and Symons, 1987). For example, a large number of ribozymesaccelerate phosphoester transfer reactions with a high degree ofspecificity, often cleaving only one of several phosphoesters in anoligonucleotide substrate (Cech et al., 1981; Michel and Westhof, 1990;Reinhold-Hurek and Shub, 1992). This specificity has been attributed tothe requirement that the substrate bind via specific base-pairinginteractions to the internal guide sequence (“IGS”) of the ribozymeprior to chemical reaction.

Ribozyme catalysis has primarily been observed as part ofsequence-specific cleavage/ligation reactions involving nucleic acids(Joyce, 1989; Cech et al., 1981). For example, U.S. Pat. No. 5,354,855(specifically incorporated herein by reference) reports that certainribozymes can act as endonucleases with a sequence specificity greaterthan that of known ribonucleases and approaching that of the DNArestriction enzymes. Thus, sequence-specific ribozyme-mediatedinhibition of gene expression may be particularly suited to therapeuticapplications (Scanlon et al., 1991; Sarver et al., 1990). Recently, itwas reported that ribozymes elicited genetic changes in some cells linesto which they were applied; the altered genes included the oncogenesH-ras, c-fos and genes of HIV. Most of this work involved themodification of a target mRNA, based on a specific mutant codon that iscleaved by a specific ribozyme.

Six basic varieties of naturally-occurring enzymatic RNAs are knownpresently. Each can catalyze the hydrolysis of RNA phosphodiester bondsin trans (and thus can cleave other RNA molecules) under physiologicalconditions. In general, enzymatic nucleic acids act by first binding toa target RNA. Such binding occurs through the target binding portion ofa enzymatic nucleic acid which is held in close proximity to anenzymatic portion of the molecule that acts to cleave the target RNA.Thus, the enzymatic nucleic acid first recognizes and then binds atarget RNA through complementary base-pairing, and once bound to thecorrect site, acts enzymatically to cut the target RNA. Strategiccleavage of such a target RNA will destroy its ability to directsynthesis of an encoded protein. After an enzymatic nucleic acid hasbound and cleaved its RNA target, it is released from that RNA to searchfor another target and can repeatedly bind and cleave new targets.

The enzymatic nature of a ribozyme is advantageous over manytechnologies, such as antisense technology (where a nucleic acidmolecule simply binds to a nucleic acid target to block its translation)since the concentration of ribozyme necessary to affect a therapeutictreatment is lower than that of an antisense oligonucleotide. Thisadvantage reflects the ability of the ribozyme to act enzymatically.Thus, a single ribozyme molecule is able to cleave many molecules oftarget RNA. In addition, the ribozyme is a highly specific inhibitor,with the specificity of inhibition depending not only on the basepairing mechanism of binding to the target RNA, but also on themechanism of target RNA cleavage. Single mismatches, orbase-substitutions, near the site of cleavage can completely eliminatecatalytic activity of a ribozyme. Similar mismatches in antisensemolecules do not prevent their action (Woolf et al., 1992). Thus, thespecificity of action of a ribozyme is greater than that of an antisenseoligonucleotide binding the same RNA site.

The enzymatic nucleic acid molecule may be formed in a hammerhead,hairpin, a hepatitis δ virus, group I intron or RNaseP RNA (inassociation with an RNA guide sequence) or Neurospora VS RNA motif.Examples of hammerhead motifs are described by Rossi et al. (1992).Examples of hairpin motifs are described by Hampel et al. (Eur. Pat.Appl. Publ. No. EP 0360257), Hampel and Tritz (1989), Hampel et al.(1990) and U.S. Pat. No. 5,631,359 (specifically incorporated herein byreference). An example of the hepatitis δ virus motif is described byPerrotta and Been (1992); an example of the RNaseP motif is described byGuerrier-Takada et al. (1983); Neurospora VS RNA ribozyme motif isdescribed by Collins (Saville and Collins, 1990; Saville and Collins,1991; Collins and Olive, 1993); and an example of the Group I intron isdescribed in (U.S. Pat. No. 4,987,071, specifically incorporated hereinby reference). All that is important in an enzymatic nucleic acidmolecule of this invention is that it has a specific substrate bindingsite which is complementary to one or more of the target gene RNAregions, and that it have nucleotide sequences within or surroundingthat substrate binding site which impart an RNA cleaving activity to themolecule. Thus the ribozyme constructs need not be limited to specificmotifs mentioned herein.

In certain embodiments, it may be important to produce enzymaticcleaving agents which exhibit a high degree of specificity for the RNAof a desired target, such as one of the sequences disclosed herein. Theenzymatic nucleic acid molecule is preferably targeted to a highlyconserved sequence region of a target mRNA. Such enzymatic nucleic acidmolecules can be delivered exogenously to specific cells as required.Alternatively, the ribozymes can be expressed from DNA or RNA vectorsthat are delivered to specific cells.

Small enzymatic nucleic acid motifs (e.g., of the hammerhead or thehairpin structure) may also be used for exogenous delivery. The simplestructure of these molecules increases the ability of the enzymaticnucleic acid to invade targeted regions of the mRNA structure.Alternatively, catalytic RNA molecules can be expressed within cellsfrom eukaryotic promoters (e.g., Scanlon et al., 1991; Kashani-Sabet etal., 1992; Dropulic et al., 1992; Weerasinghe et al., 1991; Ojwang etal., 1992; Chen et al., 1992; Sarver et al., 1990). Those skilled in theart realize that any ribozyme can be expressed in eukaryotic cells fromthe appropriate DNA vector. The activity of such ribozymes can beaugmented by their release from the primary transcript by a secondribozyme (Int. Pat. Appl. Publ. No. WO 93/23569, and Int. Pat. Appl.Publ. No. WO 94/02595, both hereby incorporated by reference; Ohkawa etal., 1992; Taira et al., 1991; and Ventura et al., 1993).

Ribozymes may be added directly, or can be complexed with cationiclipids, lipid complexes, packaged within liposomes, or otherwisedelivered to target cells. The RNA or RNA complexes can be locallyadministered to relevant tissues ex vivo, or in vivo through injection,aerosol inhalation, infusion pump or stent, with or without theirincorporation in biopolymers.

Ribozymes may be designed as described in Int. Pat. Appl. Publ. No. WO93/23569 and Int. Pat. Appl. Publ. No. WO 94/02595, each specificallyincorporated herein by reference) and synthesized to be tested in vitroand in vivo, as described. Such ribozymes can also be optimized fordelivery. While specific examples are provided, those in the art willrecognize that equivalent RNA targets in other species can be utilizedwhen necessary.

Hammerhead or hairpin ribozymes may be individually analyzed by computerfolding (Jaeger et al., 1989) to assess whether the ribozyme sequencesfold into the appropriate secondary structure. Those ribozymes withunfavorable intramolecular interactions between the binding arms and thecatalytic core are eliminated from consideration. Varying binding armlengths can be chosen to optimize activity. Generally, at least 5 or sobases on each arm are able to bind to, or otherwise interact with, thetarget RNA.

Ribozymes of the hammerhead or hairpin motif may be designed to annealto various sites in the mRNA message, and can be chemically synthesized.The method of synthesis used follows the procedure for normal RNAsynthesis as described in Usman et al. (1987) and in Scaringe et al.(1990) and makes use of common nucleic acid protecting and couplinggroups, such as dimethoxytrityl at the 5′-end, and phosphoramidites atthe 3′-end. Average stepwise coupling yields are typically >98%. Hairpinribozymes may be synthesized in two parts and annealed to reconstruct anactive ribozyme (Chowrira and Burke, 1992). Ribozymes may be modifiedextensively to enhance stability by modification with nuclease resistantgroups, for example, 2′-amino, 2′-C-allyl, 2′-flouro, 2′-o-methyl, 2′-H(for a review see e.g., Usman and Cedergren, 1992). Ribozymes may bepurified by gel electrophoresis using general methods or by highpressure liquid chromatography and resuspended in water.

Ribozyme activity can be optimized by altering the length of theribozyme binding arms, or chemically synthesizing ribozymes withmodifications that prevent their degradation by serum ribonucleases (seee.g., Int. Pat. Appl. Publ. No. WO 92/07065; Perrault et al, 1990;Pieken et al., 1991; Usman and Cedergren, 1992; Int. Pat. Appl. Publ.No. WO 93/15187; Int. Pat. Appl. Publ. No. WO 91/03162; Eur. Pat. Appl.Publ. No. 92110298.4; U.S. Pat. No. 5,334,711; and Int. Pat. Appl. Publ.No. WO 94/13688, which describe various chemical modifications that canbe made to the sugar moieties of enzymatic RNA molecules), modificationswhich enhance their efficacy in cells, and removal of stem II bases toshorten RNA synthesis times and reduce chemical requirements.

Sullivan et al. (Int. Pat. Appl. Publ. No. WO 94/02595) describes thegeneral methods for delivery of enzymatic RNA molecules. Ribozymes maybe administered to cells by a variety of methods known to those familiarto the art, including, but not restricted to, encapsulation inliposomes, by iontophoresis, or by incorporation into other vehicles,such as hydrogels, cyclodextrins, biodegradable nanocapsules, andbioadhesive microspheres. For some indications, ribozymes may bedirectly delivered ex vivo to cells or tissues with or without theaforementioned vehicles. Alternatively, the RNA/vehicle combination maybe locally delivered by direct inhalation, by direct injection or by useof a catheter, infusion pump or stent. Other routes of delivery include,but are not limited to, intravascular, intramuscular, subcutaneous orjoint injection, aerosol inhalation, oral (tablet or pill form),topical, systemic, ocular, intraperitoneal and/or intrathecal delivery.More detailed descriptions of ribozyme delivery and administration areprovided in Int. Pat. Appl. Publ. No. WO 94/02595 and Int. Pat. Appl.Publ. No. WO 93/23569, each specifically incorporated herein byreference.

Another means of accumulating high concentrations of a ribozyme(s)within cells is to incorporate the ribozyme-encoding sequences into aDNA expression vector. Transcription of the ribozyme sequences aredriven from a promoter for eukaryotic RNA polymerase I (pol I), RNApolymerase II (pol II), or RNA polymerase III (pol III). Transcriptsfrom pol II or pol III promoters will be expressed at high levels in allcells; the levels of a given pol II promoter in a given cell type willdepend on the nature of the gene regulatory sequences (enhancers,silencers, etc.) present nearby. Prokaryotic RNA polymerase promotersmay also be used, providing that the prokaryotic RNA polymerase enzymeis expressed in the appropriate cells (Elroy-Stein and Moss, 1990; Gaoand Huang, 1993; Lieber et al., 1993; Zhou et al., 1990). Ribozymesexpressed from such promoters can function in mammalian cells (e.g.Kashani-Saber et al., 1992; Ojwang et al., 1992; Chen et al., 1992; Yuet al., 1993; L'Huillier et al., 1992; Lisziewicz et al., 1993). Suchtranscription units can be incorporated into a variety of vectors forintroduction into mammalian cells, including but not restricted to,plasmid DNA vectors, viral DNA vectors (such as adenovirus oradeno-associated vectors), or viral RNA vectors (such as retroviral,semliki forest virus, sindbis virus vectors).

Ribozymes of this invention may be used as diagnostic tools to examinegenetic drift and mutations within diseased cells. They can also be usedto assess levels of the target RNA molecule. The close relationshipbetween ribozyme activity and the structure of the target RNA allows thedetection of mutations in any region of the molecule which alters thebase-pairing and three-dimensional structure of the target RNA. By usingmultiple ribozymes described in this invention, one may map nucleotidechanges which are important to RNA structure and function in vitro, aswell as in cells and tissues. Cleavage of target RNAs with ribozymes maybe used to inhibit gene expression and define the role (essentially) ofspecified gene products in the progression of disease. In this manner,other genetic targets may be defined as important mediators of thedisease. These studies will lead to better treatment of the diseaseprogression by affording the possibility of combinational therapies(e.g., multiple ribozymes targeted to different genes, ribozymes coupledwith known small molecule inhibitors, or intermittent treatment withcombinations of ribozymes and/or other chemical or biologicalmolecules). Other in vitro uses of ribozymes of this invention are wellknown in the art, and include detection of the presence of mRNAassociated with an IL-5 related condition. Such RNA is detected bydetermining the presence of a cleavage product after treatment with aribozyme using standard methodology.

4.10 Vectors for Cloning, Gene Transfer and Expression

In certain embodiments of the invention, expression vectors are employedto express a NOEY2 or NOEY2-derived polypeptide product, which can thenbe purified and, for example, be used to vaccinate animals, or togenerate antisera or monoclonal antibodies which may be used in avariety of diagnostic and therapeutic applications. In otherembodiments, an expression vector comprising a NOEY2 or NOEY2-derivedpolynucleotide may be used in gene therapy.

Expression requires that appropriate signals be provided in the vectors,and which include various regulatory elements, such asenhancers/promoters from both viral and mammalian sources that driveexpression of the genes of interest in host cells. Elements designed tooptimize messenger RNA stability and translatability in host cells alsoare defined. The conditions for the use of a number of dominant drugselection markers for establishing permanent, stable cell clonesexpressing the products are also provided, as is an element that linksexpression of the drug selection markers to expression of thepolypeptide.

4.10.1 Regulatory Elements

Throughout this application, the term “expression construct” is meant toinclude any type of genetic construct containing a nucleic acid codingfor a gene product in which part or all of the nucleic acid encodingsequence is capable of being transcribed. Preferably, such a sequenceencodes all or part of a gene which encodes a NOEY2 polypeptide. Thetranscript may be translated into a protein, but it need not be. Incertain embodiments, expression includes both transcription of a geneand translation of mRNA into a gene product. In other embodiments,expression only includes transcription of the nucleic acid encoding agene of interest.

In preferred embodiments, the nucleic acid encoding a gene product isunder transcriptional control of a promoter. A “promoter” refers to aDNA sequence recognized by the synthetic machinery of the cell, orintroduced synthetic machinery, required to initiate the specifictranscription of a gene. The phrase “under transcriptional control”means that the promoter is in the correct location and orientation inrelation to the nucleic acid to control RNA polymerase initiation andexpression of the gene.

The term promoter will be used here to refer to a group oftranscriptional control modules that are clustered around the initiationsite for RNA polymerase II. Much of the thinking about how promoters areorganized derives from analyses of several viral promoters, includingthose for the HSV thymidine kinase (tk) and SV40 early transcriptionunits. These studies, augmented by more recent work, have shown thatpromoters are composed of discrete functional modules, each consistingof approximately 7–20 bp of DNA, and containing one or more recognitionsites for transcriptional activator or repressor proteins.

At least one module in each promoter functions to position the startsite for RNA synthesis. The best known example of this is the TATA box,but in some promoters lacking a TATA box, such as the promoter for themammalian terminal deoxynucleotidyl transferase gene and the promoterfor the SV40 late genes, a discrete element overlying the start siteitself helps to fix the place of initiation.

Additional promoter elements regulate the frequency of transcriptionalinitiation. Typically, these are located in the region 30–110 bpupstream of the start site, although a number of promoters have recentlybeen shown to contain functional elements downstream of the start siteas well. The spacing between promoter elements frequently is flexible,so that promoter function is preserved when elements are inverted ormoved relative to one another. In the tk promoter, the spacing betweenpromoter elements can be increased to 50 bp apart before activity beginsto decline. Depending on the promoter, it appears that individualelements can function either co-operatively or independently to activatetranscription.

The particular promoter employed to control the expression of a nucleicacid sequence of interest is not believed to be important, so long as itis capable of direction the expression of the nucleic acid in thetargeted cell. Thus, where a human cell is targeted, it is preferable toposition the nucleic acid coding region adjacent to and under thecontrol of a promoter that is capable of being expressed in a humancell. Generally speaking, such a promoter might include either a humanor viral promoter.

In various embodiments, the human cytomegalovirus (CMV) immediate earlygene promoter, the SV40 early promoter, the Rous sarcoma virus longterminal repeat, rat insulin promoter and glyceraldehyde-3-phosphatedehydrogenase can be used to obtain high-level expression of the codingsequence of interest. The use of other viral or mammalian cellular orbacterial phage promoters which are well-known in the art to achieveexpression of a coding sequence of interest is contemplated as well,provided that the levels of expression are sufficient for a givenpurpose.

By employing a promoter with well-known properties, the level andpattern of expression of the protein of interest following transfectionor transformation can be optimized. Further, selection of a promoterthat is regulated in response to specific physiologic signals can permitinducible expression of the gene product. Tables 2 and 3 list severalelements/promoters which may be employed, in the context of the presentinvention, to regulate the expression of the gene of interest. This listis not intended to be exhaustive of all the possible elements involvedin the promotion of gene expression but, merely, to be exemplarythereof.

Enhancers are genetic elements that increase transcription from apromoter located at a distant position on the same molecule of DNA.Enhancers are organized much like promoters. That is, they are composedof many individual elements, each of which binds to one or moretranscriptional proteins.

The basic distinction between enhancers and promoters is operational. Anenhancer region as a whole must be able to stimulate transcription at adistance; this need not be true of a promoter region or its componentelements. On the other hand, a promoter must have one or more elementsthat direct initiation of RNA synthesis at a particular site and in aparticular orientation, whereas enhancers lack these specificities.Promoters and enhancers are often overlapping and contiguous, oftenseeming to have a very similar modular organization.

Where a cDNA insert is employed, one will typically desire to include apolyadenylation signal to effect proper polyadenylation of the genetranscript. The nature of the polyadenylation signal is not believed tobe crucial to the successful practice of the invention, and any suchsequence may be employed such as human growth hormone and SV40polyadenylation signals. Also contemplated as an element of theexpression cassette is a terminator. These elements can serve to enhancemessage levels and to minimize read through from the cassette into othersequences.

4.10.2 Selectable Markers

In certain embodiments of the invention, a host cell transformed withone or more NOEY2 nucleic acid segments may be identified in vitro or invivo by including a “marker” or “reporter” gene in the expressionconstruct and/or vector which comprises the NOEY2 polynucleotide. Suchreporter or marker would confer an identifiable change to the cellpermitting easy identification of cells containing the expressionconstruct. For example, the inclusion of a drug selection marker aids incloning and in the selection of transformants. Genes that conferresistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin andhistidinol are often employed as selectable markers. Alternatively,enzymes such as herpes simplex virus thymidine kinase (tk) orchloramphenicol acetyltransferase (CAT) may also be employed, as well asone or more immunologic markers.

The selectable marker employed is not believed to be important, so longas it is capable of being expressed simultaneously with the nucleic acidencoding a gene product. Further examples of selectable markers are wellknown to one of skill in the art.

4.10.3 Multigene Constructs and IRES

In certain embodiments of the invention, the use of internal ribosomebinding sites (IRES) elements are used to create multigene, orpolycistronic, messages. IRES elements are able to bypass the ribosomescanning model of 5′ methylated Cap dependent translation and begintranslation at internal sites (Pelletier and Sonenberg, 1988). IRESelements from two members of the picornavirus family (polio andencephalomyocarditis) have been described (Pelletier and Sonenberg,1988), as well an IRES from a mammalian message (Macejak and Sarnow,1991). IRES elements can be linked to heterologous open reading frames.Multiple open reading frames can be transcribed together, each separatedby an IRES, creating polycistronic messages. By virtue of the IRESelement, each open reading frame is accessible to ribosomes forefficient translation. Multiple genes can be efficiently expressed usinga single promoter/enhancer to transcribe a single message.

Any heterologous open reading frame can be linked to IRES elements. Thisincludes genes for secreted proteins, multi-subunit proteins, encoded byindependent genes, intracellular or membrane-bound proteins andselectable markers. In this way, expression of several proteins can besimultaneously engineered into a cell with a single construct and asingle selectable marker.

4.10.4 Delivery of Expression Vectors

There are a number of ways in which expression vectors may introducedinto cells. In certain embodiments of the invention, the expressionconstruct comprises a virus or engineered construct derived from a viralgenome. The ability of certain viruses to enter cells viareceptor-mediated endocytosis, to integrate into host cell genome andexpress viral genes stably and efficiently have made them attractivecandidates for the transfer of foreign genes into mammalian cells(Ridgeway, 1988; Nicolas and Rubenstein, 1988; Baichwal and Sugden,1986; Temin, 1986). The first viruses used as gene vectors were DNAviruses including the papovaviruses (simian virus 40, bovine papillomavirus, and polyoma) (Ridgeway, 1988; Baichwal and Sugden, 1986) andadenoviruses (Ridgeway, 1988; Baichwal and Sugden, 1986). These have arelatively low capacity for foreign DNA sequences and have a restrictedhost spectrum. Furthermore, their oncogenic potential and cytopathiceffects in permissive cells raise safety concerns. They can accommodateonly up to 8 kb of foreign genetic material but can be readilyintroduced in a variety of cell lines and laboratory animals (Nicolasand Rubenstein, 1988; Temin, 1986).

One of the preferred methods for in vivo delivery involves the use of anadenovirus expression vector. “Adenovirus expression vector” is meant toinclude those constructs containing adenovirus sequences sufficient to(a) support packaging of the construct and (b) to express an antisensepolynucleotide that has been cloned therein. In this context, expressiondoes not require that the gene product be synthesized.

The expression vector comprises a genetically engineered form ofadenovirus. Knowledge of the genetic organization of adenovirus, a 36kb, linear, double-stranded DNA virus, allows substitution of largepieces of adenoviral DNA with foreign sequences up to 7 kb (Grunhaus andHorwitz, 1992). In contrast to retrovirus, the adenoviral infection ofhost cells does not result in chromosomal integration because adenoviralDNA can replicate in an episomal manner without potential genotoxicity.Also, adenoviruses are structurally stable, and no genome rearrangementhas been detected after extensive amplification. Adenovirus can infectvirtually all epithelial cells regardless of their cell cycle stage. Sofar, adenoviral infection appears to be linked only to mild disease suchas acute respiratory disease in humans.

Adenovirus is particularly suitable for use as a gene transfer vectorbecause of its mid-sized genome, ease of manipulation, high titer, widetarget cell range and high infectivity. Generation and propagation ofadenovirus vectors, which are replication deficient, depend on a uniquehelper cell line, designated 293, which was transformed from humanembryonic kidney cells by Ad5 DNA fragments and constitutively expressesE1 proteins (Graham et al., 1977). Since the E3 region is dispensablefrom the adenovirus genome (Jones and Shenk, 1978), the currentadenovirus vectors, with the help of 293 cells, carry foreign DNA ineither the E1, the D3 or both regions (Graham and Prevec, 1991). Innature, adenovirus can package approximately 105% of the wild-typegenome (Ghosh-Choudhury et al., 1987), providing capacity for about 2extra kb of DNA. Combined with the approximately 5.5 kb of DNA that isreplaceable in the E1 and E3 regions, the maximum capacity of thecurrent adenovirus vector is under 7.5 kb, or about 15% of the totallength of the vector. More than 80% of the adenovirus viral genomeremains in the vector backbone and is the source of vector-bornecytotoxicity. Also, the replication deficiency of the E1-deleted virusis incomplete. For example, leakage of viral gene expression has beenobserved with the currently available vectors at high multiplicities ofinfection (MOI) (Rich et al. 1993).

Helper cell lines may be derived from human cells such as humanembryonic kidney cells, muscle cells, hematopoietic cells or other humanembryonic mesenchymal or epithelial cells. Alternatively, the helpercells may be derived from the cells of other mammalian species that arepermissive for human adenovirus. Such cells include, e.g., Vero cells orother monkey embryonic mesenchymal or epithelial cells. As stated above,the preferred helper cell line is 293.

Methods for culturing 293 cells and propagating adenovirus have beendescribed. In one format, natural cell aggregates are grown byinoculating individual cells into 1 liter siliconized spinner flasks(Techne, Cambridge, UK) containing 100–200 ml of medium. Followingstirring at 40 rpm, the cell viability is estimated with trypan blue. Inanother format, Fibra-Cel microcarriers (Bibby Sterlin, Stone, UK) (5g/l) is employed as follows. A cell inoculum, resuspended in 5 ml ofmedium, is added to the carrier (50 ml) in a 250 ml Erlenmeyer flask andleft stationary, with occasional agitation, for 1 to 4 h. The medium isthen replaced with 50 ml of fresh medium and shaking initiated. Forvirus production, cells are allowed to grow to about 80% confluence,after which time the medium is replaced (to 25% of the final volume) andadenovirus added at an MOI of 0.05. Cultures are left stationaryovernight, following which the volume is increased to 100% and shakingcommenced for another 72 h.

Adenovirus is easy to grow and manipulate and exhibits broad host rangein vitro and in vivo. This group of viruses can be obtained in hightiters, e.g., 10⁹–10¹¹ plaque-forming units per ml, and they are highlyinfective. The life cycle of adenovirus does not require integrationinto the host cell genome. The foreign genes delivered by adenovirusvectors are episomal and, therefore, have low genotoxicity to hostcells. No side effects have been reported in studies of vaccination withwild-type adenovirus (Couch et al., 1963; Top et al., 1971),demonstrating their safety and therapeutic potential as in vivo genetransfer vectors.

Adenovirus vectors have been used in eukaryotic gene expression (Levreroet al., 1991; Gomez-Foix et al., 1992) and vaccine development (Grunhausand Horwitz, 1992; Graham and Prevec, 1992). Recently, animal studiessuggested that recombinant adenovirus could be used for gene therapy(Stratford-Perricaudet and Perricaudet, 1991; Stratford-Perricaudet etal., 1990; Rich et al., 1993). Studies in administering recombinantadenovirus to different tissues include trachea instillation (Rosenfeldet al., 1991; Rosenfeld et al., 1992), muscle injection (Ragot et al.,1993), peripheral intravenous injections (Herz and Gerard, 1993) andstereotactic inoculation into the brain (Le Gal La Salle et al., 1993).

The retroviruses are a group of single-stranded RNA virusescharacterized by an ability to convert their RNA to double-stranded DNAin infected cells by a process of reverse-transcription (Coffin, 1990).The resulting DNA then stably integrates into cellular chromosomes as aprovirus and directs synthesis of viral proteins. The integrationresults in the retention of the viral gene sequences in the recipientcell and its descendants. The retroviral genome contains three genes,gag, pol, and env that code for capsid proteins, polymerase enzyme, andenvelope components, respectively. A sequence found upstream from thegag gene contains a signal for packaging of the genome into virions. Twolong terminal repeat (LTR) sequences are present at the 5′ and 3′ endsof the viral genome. These contain strong promoter and enhancersequences and are also required for integration in the host cell genome(Coffin, 1990).

In order to construct a retroviral vector, a nucleic acid encoding agene of interest is inserted into the viral genome in the place ofcertain viral sequences to produce a virus that isreplication-defective. In order to produce virions, a packaging cellline containing the gag, pol, and env genes but without the LTR andpackaging components is constructed (Mann et al., 1983). When arecombinant plasmid containing a cDNA, together with the retroviral LTRand packaging sequences is introduced into this cell line (by calciumphosphate precipitation for example), the packaging sequence allows theRNA transcript of the recombinant plasmid to be packaged into viralparticles, which are then secreted into the culture media (Nicolas andRubenstein, 1988; Temin, 1986; Mann et al., 1983). The media containingthe recombinant retroviruses is then collected, optionally concentrated,and used for gene transfer. Retroviral vectors are able to infect abroad variety of cell types. However, integration and stable expressionrequire the division of host cells (Paskind et al., 1975). A novelapproach designed to allow specific targeting of retrovirus vectors wasrecently developed based on the chemical modification of a retrovirus bythe chemical addition of lactose residues to the viral envelope. Thismodification could permit the specific infection of hepatocytes viasialoglycoprotein receptors.

A different approach to targeting of recombinant retroviruses wasdesigned in which biotinylated antibodies against a retroviral envelopeprotein and against a specific cell receptor were used. The antibodieswere coupled via the biotin components by using streptavidin (Roux etal., 1989). Using antibodies against major histocompatibility complexclass I and class II antigens, they demonstrated the infection of avariety of human cells that bore those surface antigens with anecotropic virus in vitro (Roux et al., 1989).

There are certain limitations to the use of retrovirus vectors in allaspects of the present invention. For example, retrovirus vectorsusually integrate into random sites in the cell genome. This can lead toinsertional mutagenesis through the interruption of host genes orthrough the insertion of viral regulatory sequences that can interferewith the function of flanking genes (Varmus et al., 1981). Anotherconcern with the use of defective retrovirus vectors is the potentialappearance of wild-type replication-competent virus in the packagingcells. This can result from recombination events in which the intact-sequence from the recombinant virus inserts upstream from the gag, pol,env sequence integrated in the host cell genome. However, new packagingcell lines are now available that should greatly decrease the likelihoodof recombination (Markowitz et al., 1988; Hersdorffer et al., 1990).

Other viral vectors may be employed as expression constructs in thepresent invention. Vectors derived from viruses such as vaccinia virus(Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988)adeno-associated virus (AAV) (Ridgeway, 1988; Baichwal and Sugden, 1986;Hermonat and Muzycska, 1984) and herpesviruses may be employed. Theyoffer several attractive features for various mammalian cells(Friedmann, 1989; Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar etal., 1988; Horwich et al., 1990).

With the recent recognition of defective hepatitis B viruses, newinsight was gained into the structure-function relationship of differentviral sequences. In vitro studies showed that the virus could retain theability for helper-dependent packaging and reverse transcription despitethe deletion of up to 80% of its genome (Horwich et al., 1990). Thissuggested that large portions of the genome could be replaced withforeign genetic material. The hepatotropism and persistence(integration) were particularly attractive properties for liver-directedgene transfer. Chang et al., recently introduced the chloramphenicolacetyltransferase (CAT) gene into duck hepatitis B virus genome in theplace of the polymerase, surface, and pre-surface coding sequences. Itwas co-transfected with wild-type virus into an avian hepatoma cellline. Culture media containing high titers of the recombinant virus wereused to infect primary duckling hepatocytes. Stable CAT gene expressionwas detected for at least 24 days after transfection (Chang et al.,1991).

In order to effect expression of sense or antisense gene constructs, theexpression construct must be delivered into a cell. This delivery may beaccomplished in vitro, as in laboratory procedures for transformingcells lines, or in vivo or ex vivo, as in the treatment of certaindisease states. One mechanism for delivery is via viral infection wherethe expression construct is encapsidated in an infectious viralparticle.

Several non-viral methods for the transfer of expression constructs intocultured mammalian cells also are contemplated by the present invention.These include calcium phosphate precipitation (Graham and Van Der Eb,1973; Chen and Okayama, 1987; Rippe et al., 1990) DEAE-dextran (Gopal,1985), electroporation (Tur-Kaspa et al., 1986; Potter et al., 1984),direct microinjection (Harland and Weintraub, 1985), DNA-loadedliposomes (Nicolau and Sene, 1982; Fraley et al., 1979) andlipofectamine-DNA complexes, cell sonication (Fechheimer et al., 1987),gene bombardment using high velocity microprojectiles (Yang et al.,1990), and receptor-mediated transfection (Wu and Wu, 1987; Wu and Wu,1988). Some of these techniques may be successfully adapted for in vivoor ex vivo use.

Once the expression construct has been delivered into the cell thenucleic acid encoding the gene of interest may be positioned andexpressed at different sites. In certain embodiments, the nucleic acidencoding the gene may be stably integrated into the genome of the cell.This integration may be in the cognate location and orientation viahomologous recombination (gene replacement) or it may be integrated in arandom, non-specific location (gene augmentation). In yet furtherembodiments, the nucleic acid may be stably maintained in the cell as aseparate, episomal segment of DNA. Such nucleic acid segments or“episomes” encode sequences sufficient to permit maintenance andreplication independent of or in synchronization with the host cellcycle. How the expression construct is delivered to a cell and where inthe cell the nucleic acid remains is dependent on the type of expressionconstruct employed.

In yet another embodiment of the invention, the expression construct maysimply consist of naked recombinant DNA or plasmids. Transfer of theconstruct may be performed by any of the methods mentioned above whichphysically or chemically permeabilize the cell membrane. This isparticularly applicable for transfer in vitro but it may be applied toin vivo use as well. Dubensky et al. (1984) successfully injectedpolyomavirus DNA in the form of calcium phosphate precipitates intoliver and spleen of adult and newborn mice demonstrating active viralreplication and acute infection. Benvenisty and Neshif (1986) alsodemonstrated that direct intraperitoneal injection of calciumphosphate-precipitated plasmids results in expression of the transfectedgenes. It is envisioned that DNA encoding a gene of interest may also betransferred in a similar manner in vivo and express the gene product.

In still another embodiment of the invention for transferring a nakedDNA expression construct into cells may involve particle bombardment.This method depends on the ability to accelerate DNA-coatedmicroprojectiles to a high velocity allowing them to pierce cellmembranes and enter cells without killing them (Klein et al., 1987).Several devices for accelerating small particles have been developed.One such device relies on a high voltage discharge to generate anelectrical current, which in turn provides the motive force (Yang etal., 1990). The microprojectiles used have consisted of biologicallyinert substances such as tungsten or gold beads.

Selected organs including the liver, skin, and muscle tissue of rats andmice have been bombarded in vivo (Yang et al., 1990; Zelenin et al.,1991). This may require surgical exposure of the tissue or cells, toeliminate any intervening tissue between the gun and the target organ,i.e., ex vivo treatment. Again, DNA encoding a particular gene may bedelivered via this method and still be incorporated by the presentinvention.

In one embodiment, such expression constructs may be entrapped in aliposome, lipid complex, nanocapsule, or other formulation using one ormore of the methods disclosed in Section 4.8. Also contemplated arelipofectamine-DNA complexes. For example, liposome-mediated nucleic aciddelivery and expression of foreign DNA in vitro has been verysuccessful. Wong et al. (1980) demonstrated the feasibility ofliposome-mediated delivery and expression of foreign DNA in culturedchick embryo, HeLa and hepatoma cells. Nicolau et al. (1987)accomplished successful liposome-mediated gene transfer in rats afterintravenous injection.

In certain embodiments of the invention, the liposome may be complexedwith a hemagglutinating virus (HVJ). This has been shown to facilitatefusion with the cell membrane and promote cell entry ofliposome-encapsulated DNA (Kaneda et al., 1989). In other embodiments,the liposome may be complexed or employed in conjunction with nuclearnon-histone chromosomal proteins (HMG-1) (Kato et al., 1991). In yetfurther embodiments, the liposome may be complexed or employed inconjunction with both HVJ and HMG-1. In that such expression constructshave been successfully employed in transfer and expression of nucleicacid in vitro and in vivo, then they are applicable for the presentinvention. Where a bacterial promoter is employed in the DNA construct,it also will be desirable to include within the liposome an appropriatebacterial polymerase.

Other expression constructs which can be employed to deliver a nucleicacid encoding a particular gene into cells are receptor-mediateddelivery vehicles. These take advantage of the selective uptake ofmacromolecules by receptor-mediated endocytosis in almost all eukaryoticcells. Because of the cell type-specific distribution of variousreceptors, the delivery can be highly specific (Wu and Wu, 1993).

Receptor-mediated gene targeting vehicles generally consist of twocomponents: a cell receptor-specific ligand and a DNA-binding agent.Several ligands have been used for receptor-mediated gene transfer. Themost extensively characterized ligands are asialoorosomucoid (ASOR) (Wuand Wu, 1987) and transferrin (Wagner et al., 1990). Recently, asynthetic neoglycoprotein, which recognizes the same receptor as ASOR,has been used as a gene delivery vehicle (Ferkol et al., 1993; Peraleset al., 1994) and epidermal growth factor (EGF) has also been used todeliver genes to squamous carcinoma cells (Eur. Pat. Appl. Publ. No. EP0360257, specifically incorporated herein by reference).

In other embodiments, the delivery vehicle may comprise a ligand and aliposome. For example, Nicolau et al., (1987) employedlactosyl-ceramide, a galactose-terminal asialganglioside, incorporatedinto liposomes and observed an increase in the uptake of the insulingene by hepatocytes. Thus, it is feasible that a nucleic acid encoding aparticular gene also may be specifically delivered into a cell type suchas lung, epithelial or tumor cells, by any number of receptor-ligandsystems with or without liposomes. For example, epidermal growth factor(EGF) may be used as the receptor for mediated delivery of a nucleicacid encoding a gene in many tumor cells that exhibit upregulation ofEGF receptor. Mannose can be used to target the mannose receptor onliver cells. Also, antibodies to CD5 (CLL), CD22 (lymphoma), CD25(T-cell leukemia) and MAA (melanoma) can similarly be used as targetingmoieties.

In certain embodiments, gene transfer may more easily be performed underex vivo conditions. Ex vivo gene therapy refers to the isolation ofcells from an animal, the delivery of a nucleic acid into the cells invitro, and then the return of the modified cells back into an animal.This may involve the surgical removal of tissue/organs from an animal orthe primary culture of cells and tissues.

Primary mammalian cell cultures may be prepared in various ways. Inorder for the cells to be kept viable while in vitro and in contact withthe expression construct, it is necessary to ensure that the cellsmaintain contact with the correct ratio of oxygen and carbon dioxide andnutrients but are protected from microbial contamination. Cell culturetechniques are well documented and are disclosed herein by reference(Freshner, 1992).

One embodiment of the foregoing involves the use of gene transfer toimmortalize cells for the production of proteins. The gene for theprotein of interest may be transferred as described above intoappropriate host cells followed by culture of cells under theappropriate conditions. The gene for virtually any polypeptide may beemployed in this manner. The generation of recombinant expressionvectors, and the elements included therein, are discussed above.Alternatively, the protein to be produced may be an endogenous proteinnormally synthesized by the cell in question.

Examples of useful mammalian host cell lines are Vero and HeLa cells andcell lines of Chinese hamster ovary, W138, BHK, COS-7, 293, HepG2,NIH3T3, RIN and MDCK cells. In addition, a host cell strain may bechosen that modulates the expression of the inserted sequences, ormodifies and process the gene product in the manner desired. Suchmodifications (e.g., glycosylation) and processing (e.g., cleavage) ofprotein products may be important for the function of the protein.Different host cells have characteristic and specific mechanisms for thepost-translational processing and modification of proteins. Appropriatecell lines or host systems can be chosen to insure the correctmodification and processing of the foreign protein expressed.

A number of selection systems may be used including, but not limited to,HSV thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase andadenine phosphoribosyltransferase genes, in tk-, hgprt- or aprt- cells,respectively. Also, anti-metabolite resistance can be used as the basisof selection for dhfr, that confers resistance to; gpt, that confersresistance to mycophenolic acid; neo, that confers resistance to theaminoglycoside G418; and hygro, that confers resistance to hygromycin.

Animal cells can be propagated in vitro in two modes: as non-anchoragedependent cells growing in suspension throughout the bulk of the cultureor as anchorage-dependent cells requiring attachment to a solidsubstrate for their propagation (i.e. a monolayer type of cell growth).

Non-anchorage dependent or suspension cultures from continuousestablished cell lines are the most widely used means of large scaleproduction of cells and cell products. However, suspension culturedcells have limitations, such as tumorigenic potential and lower proteinproduction than adherent T-cells.

Large scale suspension culture of mammalian cells in stirred tanks is acommon method for production of recombinant proteins. Two suspensionculture reactor designs are in wide use—the stirred reactor and theairlift reactor. The stirred design has successfully been used on an8000 liter capacity for the production of interferon. Cells are grown ina stainless steel tank with a height-to-diameter ratio of 1:1 to 3:1.The culture is usually mixed with one or more agitators, based on bladeddisks or marine propeller patterns. Agitator systems offering less shearforces than blades have been described. Agitation may be driven eitherdirectly or indirectly by magnetically coupled drives. Indirect drivesreduce the risk of microbial contamination through seals on stirrershafts.

The airlift reactor, also initially described for microbial fermentationand later adapted for mammalian culture, relies on a gas stream to bothmix and oxygenate the culture. The gas stream enters a riser section ofthe reactor and drives circulation. Gas disengages at the culturesurface, causing denser liquid free of gas bubbles to travel downward inthe downcomer section of the reactor. The main advantage of this designis the simplicity and lack of need for mechanical mixing. Typically, theheight-to-diameter ratio is 10:1. The airlift reactor scales uprelatively easily, has good mass transfer of gases and generatesrelatively low shear forces.

4.11 Liposomes and Nanocapsules

In certain embodiments, the inventors contemplate the use of liposomesand/or nanocapsules for the introduction of a NOEY2 composition into ahost cell. Such formulations may be preferred for the introduction ofpharmaceutically-acceptable formulations of the polypeptides,pharmaceuticals, and/or antibodies disclosed herein. The formation anduse of liposomes is generally known to those of skill in the art (seefor example, Couvreur et al., 1977 which describes the use of liposomesand nanocapsules in the targeted antibiotic therapy of intracellularbacterial infections and diseases). More recently, liposomes weredeveloped with improved serum stability and circulation half-times(Gabizon and Papahadjopoulos, 1988; Allen and Choun, 1987).

In one instance, the disclosed composition may be entrapped in aliposome. Liposomes are vesicular structures characterized by aphospholipid bilayer membrane and an inner aqueous medium. Multilamellarliposomes have multiple lipid layers separated by aqueous medium. Theterm “liposome” is intended to mean a composition arising spontaneouslywhen phospholipids are suspended in an excess of aqueous solution. Thelipid components undergo self-rearrangement before the formation ofclosed structures and entrap water and dissolved solutes between thelipid bilayers (Ghosh and Bachhawat, 1991).

Nanocapsules can generally entrap compounds in a stable and reproducibleway (Henry-Michelland et al., 1987). To avoid side effects due tointracellular polymeric overloading, such ultrafine particles (sizedaround 0.1 μm) should be designed using polymers able to be degraded invivo. Biodegradable polyalkyl-cyano-acrylate nanoparticles that meetthese requirements are contemplated for use in the present invention,and such particles may be are easily made, as described (Couvreur etal., 1977; 1988). Methods of preparing polyalkyl-cyano-acrylatenanoparticles containing biologically active substances and their useare described in U.S. Pat. No. 4,329,332, U.S. Pat. No. 4,489,055, andU.S. Pat. No. 4,913,908.

Pharmaceutical compositions containing nanocapsules for the oraldelivery of active agents are described in U.S. Pat. No. 5,500,224 andU.S. Pat. No. 5,620,708. U.S. Pat. No. 5,500,224 describes apharmaceutical composition in the form of a colloidal suspension ofnanocapsules comprising an oily phase consisting essentially of an oilcontaining dissolved therein a surfactant and suspended therein aplurality of nanocapsules having a diameter of less than 500 nanometers.U.S. Pat. No. 5,620,708 describes compositions and methods for the oraladministration of drugs and other active agents. The compositionscomprise an active agent carrier particle attached to a binding moietywhich binds specifically to a target molecule present on the surface ofa mammalian enterocyte. The binding moiety binds to the target moleculewith a binding affinity or avidity sufficient to initiate endocytosis orphagocytosis of the particulate active agent carrier so that the carrierwill be absorbed by the enterocyte. The active agent will then bereleased from the carrier to the host's systemic circulation. In thisway, degradation of degradation-sensitive drugs, such as polypeptides,in the intestines can be avoided while absorption of proteins andpolypeptides form the intestinal tract is increased.

U.S. Pat. No. 5,641,515 and U.S. Pat. No. 5,698,515 describe the use ofnanocapsules for the oral administration of a polypeptide, specifically,insulin and are incorporated herein by reference. U.S. Pat. No.5,698,515 described insulin containing nanocapsules intended for oraladministration of insulin which comprises a hydrophilic polymer modifiedwith an inhibitor of proteolytic enzyme, insulin and water, wherein theinhibitor of proteolytic enzymes is ovomucoid isolated from duck orturkey egg whites. U.S. Pat. No. 5,556,617 describes the use ofnanoparticles as pharmaceutical treatment of the upper epidermal layersby topical application on the skin.

Poly(alkyl cyanoacrylate) nanocapsules have been used as biodegradablepolymeric drug carriers for subcutaneous and peroral delivery ofoctreotide, a long-acting somatostatin analogue. The nanocapsules,prepared by interfacial emulsion polymerization of isobutylcyanoacrylate, were 216 nm in diameter and incorporated 60% ofoctreotide. Nanocapsules were administered subcutaneously and theoctreotide-loaded nanocapsules (20 mg/kg) suppressed the insulinaemiapeak induced by intravenous glucose overload and depressed insulinsecretion over 48 h. When administered perorally to oestrogen-treatedrats, octreotide loaded nanocapsules (200 and 100 mg/kg) significantlyimproved the reduction of prolactin secretion and slightly increasedplasma octreotide levels (Damge et al., 1997).

The negative surface charge of nanocapsules makes them particularlysusceptible to lysozyme (LZM), a positively-charged enzyme that ishighly concentrated in mucosas. This interaction causes destabilizationof the nanocapsule by LZM; however, it was observed that thedestabilizing effects caused by the adsorption of LZM onto thenanocapsules can be prevented by previous adsorption of the cationicpoly(amino acid) poly-L-lysine (Calvo et al., 1997).

Calvo et al., 1996 describe the use of poly-epsilon-caprolactone (PECL)microparticles for the ocular bioavailability of drugs. Their studyshowed that PECL nanoparticles and nanocapsules as well as submicronemulsions are shown to be novel corneal drug carriers, and represent auseful approach for increasing the ocular bioavailability of drugs.

An excellent review of nanoparticles and nanocapsular carriers isprovided by Arshady 1996. Arshady notes that one of the major obstaclesto the targeted delivery of colloidal carriers, or nanocapsules, is thebody's own defense mechanism in capturing foreign particles by thereticuloendothelial system (RES). This means that following intravenousadministration, practically all nanometer size particles are captured bythe RES (mainly the liver). The review describes recent initiatives onthe design of macromolecular homing devices which seem to disguisenanoparticles from the RES and, hence, are of potential interest to thetargeted delivery of nanocapsular carriers. The idea is based on a graftcopolymer model embodying a link site for attachment to the carrier, afloating pad for maintaining the particles afloat in the blood stream,an affinity ligand for site-specific delivery and a structural tune forbalancing the overall structure of the homing device.

Yu and Chang, 1996 describe the use of nanocapsules containinghemoglobin as potential blood substitutes. They use different polymersincluding polylactic acid and polyisobutyl-cyanoacrylate and modify thesurface of the nanocapsules with polyethylene glycol (PEG) or with PEG2000 PE. The surface modified nanocapsules containing hemoglobin survivelonger in the circulation.

U.S. Pat. No. 5,451,410 describes the use of modified amino acid for theencapsulation of active agents. Modified amino acids and methods for thepreparation and used as oral delivery systems for pharmaceutical agentsare described. The modified amino acids are preparable by reactingsingle amino acids or mixtures of two or more kinds of amino acids withan amino modifying agent such as benzene sulfonyl chloride, benzoylchloride, and hippuryl chloride. The modified amino acids formencapsulating microspheres in the presence of the active agent undersphere-forming conditions. Alternatively, the modified amino acids maybe used as a carrier by simply mixing the amino acids with the activeagent. The modified amino acids are particularly useful in deliveringpeptides, e.g., insulin or calmodulin, or other agents which aresensitive to the denaturing conditions of the gastrointestinal tract.

4.12 Diagnosing Cancers Involving NOEY2

The present inventors have determined that alterations in NOEY2 areassociated with malignancy. Therefore, a NOEY2 polypeptide or a NOEY2gene may be employed as a diagnostic or prognostic indicator of cancer.More specifically, point mutations, deletions, insertions or regulatoryperturbations relating to NOEY2 may cause cancer or promote cancerdevelopment, cause or promoter tumor progression at a primary site,and/or cause or promote metastasis. Other phenomena associated withmalignancy that may be affected by NOEY2 expression include angiogenesisand tissue invasion.

One embodiment of the instant invention comprises a method for detectingvariation in the expression of NOEY2. This may comprises determiningthat level of NOEY2 or determining specific alterations in the expressedproduct. Obviously, this sort of assay has importance in the diagnosisof related cancers. Such cancer may involve cancers of the breast orovaries, or alternatively, cancers involving the lung, liver, spleen,brain kidney, pancreas, small intestine, blood cells, lymph node, colon,endometrium, stomach, prostate, testicle, skin, head and neck,esophagus, bone marrow, blood or other tissue. In particular, thepresent invention relates to the diagnosis of breast and ovariancancers.

The biological sample can be any tissue or fluid. Various embodimentsinclude cells of the skin, muscle, facia, brain, prostate, breast,endometrium, lung, head & neck, pancreas, small intestine, blood cells,liver, testes, ovaries, colon, skin, stomach, esophagus, spleen, lymphnode, bone marrow or kidney. Other embodiments include fluid samplessuch as peripheral blood, lymph fluid, ascites, serous fluid, pleuraleffusion, sputum, cerebrospinal fluid, lacrimal fluid, stool or urine.

Nucleic acid used is isolated from cells contained in the biologicalsample, according to standard methodologies (Sambrook et al., 1989). Thenucleic acid may be genomic DNA or fractionated or whole cell RNA. WhereRNA is used, it may be desired to convert the RNA to a complementaryDNA. In one embodiment, the RNA is whole cell RNA; in another, it ispoly-A RNA. Normally, the nucleic acid is amplified.

Depending on the format, the specific nucleic acid of interest isidentified in the sample directly using amplification or with a second,known nucleic acid following amplification. Next, the identified productis detected. In certain applications, the detection may be performed byvisual means (e.g., ethidium bromide staining of a gel). Alternatively,the detection may involve indirect identification of the product viachemiluminescence, radioactive scintigraphy of radiolabel or fluorescentlabel or even via a system using electrical or thermal impulse signals(Affymax Technology; Bellus, 1994).

Following detection, one may compare the results seen in a given patientwith a statistically significant reference group of normal patients andpatients that have NOEY2-related pathologies. In this way, it ispossible to correlate the amount or kind of NOEY2 detected with variousclinical states.

Various types of defects are to be identified. Thus, “alterations”should be read as including deletions, insertions, point mutations andduplications. Point mutations result in stop codons, frameshiftmutations or amino acid substitutions. Somatic mutations are thoseoccurring in non-germline tissues. Germ-line tissue can occur in anytissue and are inherited. Mutations in and outside the coding regionalso may affect the amount of NOEY2 produced, both by altering thetranscription of the gene or in destabilizing or otherwise altering theprocessing of either the transcript (mRNA) or protein.

A variety of different assays are contemplated in this regard, includingbut not limited to, fluorescent in situ hybridization (FISH), direct DNAsequencing, PFGE analysis, Southern or Northern blotting,single-stranded conformation analysis (SSCA), RNAse protection assay,allele-specific oligonucleotide (ASO), dot blot analysis, denaturinggradient gel electrophoresis, RFLP and PCR-SSCP.

4.12.1 Primers and Probes

The term primer, as defined herein, is meant to encompass any nucleicacid that is capable of priming the synthesis of a nascent nucleic acidin a template-dependent process. Typically, primers are oligonucleotidesfrom about ten to about fifteen base pairs in length or even longersequences such as those from about twenty to about 30 base pairs or morein length, with even longer sequences be employed for certainapplications. Primers may be provided in double-stranded orsingle-stranded form, although the single-stranded form is preferred.Probes are defined differently, although they may act as primers.Probes, while perhaps capable of priming, are designed to binding to thetarget DNA or RNA and need not be used in an amplification process.

In preferred embodiments, the probes or primers are labeled withradioactive species (³²P, ¹⁴C, ³⁵S, ³H, or other label), with afluorophore (rhodamine, fluorescein) or a chemiluminescent (luciferase).

4.12.2 Template Dependent Amplification Methods

A number of template dependent processes are available to amplify themarker sequences present in a given template sample. One of the bestknown amplification methods is the polymerase chain reaction (referredto as PCR™) which is described in detail in U.S. Pat. Nos. 4,683,195,4,683,202 and 4,800,159, and in Innis et al., 1988, each of which isincorporated herein by reference in its entirety.

Briefly, in PCR™, two primer sequences are prepared that arecomplementary to regions on opposite complementary strands of the markersequence. An excess of deoxynucleoside triphosphates are added to areaction mixture along with a DNA polymerase, e.g., Taq polymerase. Ifthe marker sequence is present in a sample, the primers will bind to themarker and the polymerase will cause the primers to be extended alongthe marker sequence by adding on nucleotides. By raising and loweringthe temperature of the reaction mixture, the extended primers willdissociate from the marker to form reaction products, excess primerswill bind to the marker and to the reaction products and the process isrepeated.

A reverse transcriptase PCR™ amplification procedure (RT-PCR™) may beperformed in order to quantify the amount of mRNA amplified. Methods ofreverse transcribing RNA into cDNA are well known and described inSambrook et al., 1989. Alternative methods for reverse transcriptionutilize thermostable, RNA-dependent DNA polymerases. These methods aredescribed in WO 90/07641 filed Dec. 21, 1990. Polymerase chain reactionmethodologies are well known in the art.

Another method for amplification is the ligase chain reaction (“LCR”),disclosed in EPO No. 320 308, incorporated herein by reference in itsentirety. In LCR, two complementary probe pairs are prepared, and in thepresence of the target sequence, each pair will bind to oppositecomplementary strands of the target such that they abut. In the presenceof a ligase, the two probe pairs will link to form a single unit. Bytemperature cycling, as in PCR™, bound ligated units dissociate from thetarget and then serve as “target sequences” for ligation of excess probepairs. U.S. Pat. No. 4,883,750 describes a method similar to LCR forbinding probe pairs to a target sequence.

Qbeta Replicase, described in PCT Application No. PCT/US87/00880, mayalso be used as still another amplification method in the presentinvention. In this method, a replicative sequence of RNA that has aregion complementary to that of a target is added to a sample in thepresence of an RNA polymerase. The polymerase will copy the replicativesequence that can then be detected.

An isothermal amplification method, in which restriction endonucleasesand ligases are used to achieve the amplification of target moleculesthat contain nucleotide 5′-[α-thio]-triphosphates in one strand of arestriction site may also be useful in the amplification of nucleicacids in the present invention, Walker et al., (1992).

Strand Displacement Amplification (SDA) is another method of carryingout isothermal amplification of nucleic acids which involves multiplerounds of strand displacement and synthesis, i.e., nick translation. Asimilar method, called Repair Chain Reaction (RCR), involves annealingseveral probes throughout a region targeted for amplification, followedby a repair reaction in which only two of the four bases are present.The other two bases can be added as biotinylated derivatives for easydetection. A similar approach is used in SDA. Target specific sequencescan also be detected using a cyclic probe reaction (CPR). In CPR, aprobe having 3′ and 5′ sequences of non-specific DNA and a middlesequence of specific RNA is hybridized to DNA that is present in asample. Upon hybridization, the reaction is treated with RNase H, andthe products of the probe identified as distinctive products that arereleased after digestion. The original template is annealed to anothercycling probe and the reaction is repeated.

Still another amplification methods described in GB Application No.2,202,328, and in PCT Application No. PCT/US89/01025, each of which isincorporated herein by reference in its entirety, may be used inaccordance with the present invention. In the former application,“modified” primers are used in a PCR-like, template- andenzyme-dependent synthesis. The primers may be modified by labeling witha capture moiety (e.g., biotin) and/or a detector moiety (e.g., enzyme).In the latter application, an excess of labeled probes are added to asample. In the presence of the target sequence, the probe binds and iscleaved catalytically. After cleavage, the target sequence is releasedintact to be bound by excess probe. Cleavage of the labeled probesignals the presence of the target sequence.

Other nucleic acid amplification procedures include transcription-basedamplification systems (TAS), including nucleic acid sequence basedamplification (NASBA) and 3SR (Kwoh et al., 1989; Int. Pat. Appl. Publ.No. WO 88/10315, incorporated herein by reference in their entirety). InNASBA, the nucleic acids can be prepared for amplification by standardphenol/chloroform extraction, heat denaturation of a clinical sample,treatment with lysis buffer and minispin columns for isolation of DNAand RNA or guanidinium chloride extraction of RNA. These amplificationtechniques involve annealing a primer which has target specificsequences. Following polymerization, DNA/RNA hybrids are digested withRNase H while double stranded DNA molecules are heat denatured again. Ineither case the single stranded DNA is made fully double stranded byaddition of second target specific primer, followed by polymerization.The double-stranded DNA molecules are then multiply transcribed by anRNA polymerase such as T7 or SP6. In an isothermal cyclic reaction, theRNA's are reverse transcribed into single stranded DNA, which is thenconverted to double stranded DNA, and then transcribed once again withan RNA polymerase such as T7 or SP6. The resulting products, whethertruncated or complete, indicate target specific sequences.

Eur. Pat. Appl. Publ. No. EP 329,822 (incorporated herein by referencein its entirety) disclose a nucleic acid amplification process involvingcyclically synthesizing single-stranded RNA (“ssRNA”), ssDNA, anddouble-stranded DNA (dsDNA), which may be used in accordance with thepresent invention. The ssRNA is a template for a first primeroligonucleotide, which is elongated by reverse transcriptase(RNA-dependent DNA polymerase). The RNA is then removed from theresulting DNA:RNA duplex by the action of ribonuclease H(RNase H, anRNase specific for RNA in duplex with either DNA or RNA). The resultantssDNA is a template for a second primer, which also includes thesequences of an RNA polymerase promoter (exemplified by T7 RNApolymerase) 5′ to its homology to the template. This primer is thenextended by DNA polymerase (exemplified by the large “Klenow” fragmentof E. coli DNA polymerase I), resulting in a double-stranded DNA(“dsDNA”) molecule, having a sequence identical to that of the originalRNA between the primers and having additionally, at one end, a promotersequence. This promoter sequence can be used by the appropriate RNApolymerase to make many RNA copies of the DNA. These copies can thenre-enter the cycle leading to very swift amplification. With properchoice of enzymes, this amplification can be done isothermally withoutaddition of enzymes at each cycle. Because of the cyclical nature ofthis process, the starting sequence can be chosen to be in the form ofeither DNA or RNA.

Miller et al., PCT Application WO 89/06700 (incorporated herein byreference in its entirety) disclose a nucleic acid sequenceamplification scheme based on the hybridization of a promoter/primersequence to a target single-stranded DNA (“ssDNA”) followed bytranscription of many RNA copies of the sequence. This scheme is notcyclic, i.e., new templates are not produced from the resultant RNAtranscripts. Other amplification methods include “RACE” and “one-sidedPCR™” (Frohman, 1990; Ohara et al., 1989; each herein incorporated byreference in their entirety).

Methods based on ligation of two (or more) oligonucleotides in thepresence of nucleic acid having the sequence of the resulting“di-oligonucleotide”, thereby amplifying the di-oligonucleotide, mayalso be used in the amplification step of the present invention. Wu andWang, (1989), incorporated herein by reference in its entirety.

4.12.3 Southern/Northern Blotting

Blotting techniques are well known to those of skill in the art.Southern blotting involves the use of DNA as a target, whereas Northernblotting involves the use of RNA as a target. Each provide differenttypes of information, although cDNA blotting is analogous, in manyaspects, to blotting or RNA species.

Briefly, a probe is used to target a DNA or RNA species that has beenimmobilized on a suitable matrix, often a filter of nitrocellulose. Thedifferent species should be spatially separated to facilitate analysis.This often is accomplished by gel electrophoresis of nucleic acidspecies followed by “blotting” on to the filter.

Subsequently, the blotted target is incubated with a probe (usuallylabeled) under conditions that promote denaturation and rehybridization.Because the probe is designed to base pair with the target, the probewill binding a portion of the target sequence under renaturingconditions. Unbound probe is then removed, and detection is accomplishedas described above.

4.12.4 Separation Methods

It normally is desirable, at one stage or another, to separate theamplification product from the template and the excess primer for thepurpose of determining whether specific amplification has occurred. Inone embodiment, amplification products are separated by agarose,agarose-acrylamide or polyacrylamide gel electrophoresis using standardmethods. See Sambrook et al., 1989. Alternatively, chromatographictechniques may be employed to effect separation. There are many kinds ofchromatography which may be used in the present invention: adsorption,partition, ion-exchange and molecular sieve, and many specializedtechniques for using them including column, paper, thin-layer and gaschromatography (Freifelder et al., 1968a, Freifelder et al., 1968b;Freifelder, 1982).

4.12.5 Detection Methods

Products may be visualized in order to confirm amplification of themarker sequences. One typical visualization method involves staining ofa gel with ethidium bromide and visualization under UV light.Alternatively, if the amplification products are integrally labeled withradio- or fluorometrically-labeled nucleotides, the amplificationproducts can then be exposed to x-ray film or visualized under theappropriate stimulating spectra, following separation.

In one embodiment, visualization is achieved indirectly. Followingseparation of amplification products, a labeled nucleic acid probe isbrought into contact with the amplified marker sequence. The probepreferably is conjugated to a chromophore but may be radiolabeled. Inanother embodiment, the probe is conjugated to a binding partner, suchas an antibody or biotin, and the other member of the binding paircarries a detectable moiety.

In one embodiment, detection is by a labeled probe. The techniquesinvolved are well known to those of skill in the art and can be found inmany standard books on molecular protocols (see Sambrook et al., 1989).For example, chromophore or radiolabel probes or primers identify thetarget during or following amplification. One example of the foregoingis described in U.S. Pat. No. 5,279,721, incorporated by referenceherein, which discloses an apparatus and method for the automatedelectrophoresis and transfer of nucleic acids. The apparatus permitselectrophoresis and blotting without external manipulation of the geland is ideally suited to carrying out methods according to the presentinvention.

In addition, the amplification products described above may be subjectedto sequence analysis to identify specific kinds of variations usingstandard sequence analysis techniques. Within certain methods,exhaustive analysis of genes is carried out by sequence analysis usingprimer sets designed for optimal sequencing (Pignon et al, 1994). Thepresent invention provides methods by which any or all of these types ofanalyses may be used. Using the sequences disclosed herein,oligonucleotide primers may be designed to permit the amplification ofsequences throughout the NOEY2 gene that may then be analyzed by directsequencing.

4.12.6 Kit Components

All the essential materials and reagents required for detecting andsequencing NOEY2 and variants thereof may be assembled together in akit. This generally will comprise preselected primers and probes. Alsoincluded may be enzymes suitable for amplifying nucleic acids includingvarious polymerases (RT, Taq, Sequenase™, etc.), deoxynucleotides andbuffers to provide the necessary reaction mixture for amplification.Such kits also generally will comprise, in suitable means, distinctcontainers for each individual reagent and enzyme as well as for eachprimer or probe.

4.12.7 Relative Quantitative RT-PCR™

Reverse transcription (RT) of RNA to cDNA followed by relativequantitative PCR™ (RT-PCR™) can be used to determine the relativeconcentrations of specific mRNA species isolated from patients. Bydetermining that the concentration of a specific mRNA species varies, itis shown that the gene encoding the specific mRNA species isdifferentially expressed.

In PCR™, the number of molecules of the amplified target DNA increase bya factor approaching two with every cycle of the reaction until somereagent becomes limiting. Thereafter, the rate of amplification becomesincreasingly diminished until there is no increase in the amplifiedtarget between cycles. If a graph is plotted in which the cycle numberis on the X axis and the log of the concentration of the amplifiedtarget DNA is on the Y axis, a curved line of characteristic shape isformed by connecting the plotted points. Beginning with the first cycle,the slope of the line is positive and constant. This is said to be thelinear portion of the curve. After a reagent becomes limiting, the slopeof the line begins to decrease and eventually becomes zero. At thispoint the concentration of the amplified target DNA becomes asymptoticto some fixed value. This is said to be the plateau portion of thecurve.

The concentration of the target DNA in the linear portion of the PCR™amplification is directly proportional to the starting concentration ofthe target before the reaction began. By determining the concentrationof the amplified products of the target DNA in PCR™ reactions that havecompleted the same number of cycles and are in their linear ranges, itis possible to determine the relative concentrations of the specifictarget sequence in the original DNA mixture. If the DNA mixtures arecDNAs synthesized from RNAs isolated from different tissues or cells,the relative abundance of the specific mRNA from which the targetsequence was derived can be determined for the respective tissues orcells. This direct proportionality between the concentration of the PCR™products and the relative mRNA abundance is only true in the linearrange of the PCR™ reaction.

The final concentration of the target DNA in the plateau portion of thecurve is determined by the availability of reagents in the reaction mixand is independent of the original concentration of target DNA.Therefore, the first condition that must be met before the relativeabundance of a mRNA species can be determined by RT-PCR™ for acollection of RNA populations is that the concentrations of theamplified PCR™ products must be sampled when the PCR™ reactions are inthe linear portion of their curves.

The second condition that must be met for an RT-PCR™ experiment tosuccessfully determine the relative abundance of a particular mRNAspecies is that relative concentrations of the amplifiable cDNAs must benormalized to some independent standard. The goal of an RT-PCR™experiment is to determine the abundance of a particular mRNA speciesrelative to the average abundance of all mRNA species in the sample. Inthe studies described below, mRNAs for β-actin, asparagine synthetaseand lipocortin II were used as external and internal standards to whichthe relative abundance of other mRNAs are compared.

Most protocols for competitive PCR™ utilize internal PCR™ standards thatare approximately as abundant as the target. These strategies areeffective if the products of the PCR™ amplifications are sampled duringtheir linear phases. If the products are sampled when the reactions areapproaching the plateau phase, then the less abundant product becomesrelatively over represented. Comparisons of relative abundance made formany different RNA samples, such as is the case when examining RNAsamples for differential expression, become distorted in such a way asto make differences in relative abundance of RNAs appear less than theyactually are. This is not a significant problem if the internal standardis much more abundant than the target. If the internal standard is moreabundant than the target, then direct linear comparisons can be madebetween RNA samples.

The above discussion describes theoretical considerations for an RT-PCR™assay for clinically derived materials. The problems inherent inclinical samples are that they are of variable quantity (makingnormalization problematic), and that they are of variable quality(necessitating the co-amplification of a reliable internal control,preferably of larger size than the target). Both of these problems areovercome if the RT-PCR™ is performed as a relative quantitative RT-PCR™with an internal standard in which the internal standard is anamplifiable cDNA fragment that is larger than the target cDNA fragmentand in which the abundance of the mRNA encoding the internal standard isroughly 5–100 fold higher than the mRNA encoding the target. This assaymeasures relative abundance, not absolute abundance of the respectivemRNA species.

Other studies may be performed using a more conventional relativequantitative RT-PCR™ assay with an external standard protocol. Theseassays sample the PCR™ products in the linear portion of theiramplification curves. The number of PCR™ cycles that are optimal forsampling must be empirically determined for each target cDNA fragment.In addition, the reverse transcriptase products of each RNA populationisolated from the various tissue samples must be carefully normalizedfor equal concentrations of amplifiable cDNAs. This consideration isvery important since the assay measures absolute mRNA abundance.Absolute mRNA abundance can be used as a measure of differential geneexpression only in normalized samples. While empirical determination ofthe linear range of the amplification curve and normalization of cDNApreparations are tedious and time consuming processes, the resultingRT-PCR™ assays can be superior to those derived from the relativequantitative RT-PCR™ assay with an internal standard.

One reason for this advantage is that without the internalstandard/competitor, all of the reagents can be converted into a singlePCR™ product in the linear range of the amplification curve, thusincreasing the sensitivity of the assay. Another reason is that withonly one PCR™ product, display of the product on an electrophoretic gelor another display method becomes less complex, has less background andis easier to interpret.

4.12.8 Chip Technologies

Specifically contemplated by the present inventors are chip-based DNAtechnologies such as those described by Hacia et al. (1996) andShoemaker et al. (1996). Briefly, these techniques involve quantitativemethods for analyzing large numbers of genes rapidly and accurately. Bytagging genes with oligonucleotides or using fixed probe arrays, one canemploy chip technology to segregate target molecules as high densityarrays and screen these molecules on the basis of hybridization. Seealso Pease et al. (1994); Fodor et al. (1991).

4.13 Methods for Screening Active Compounds

The present invention also contemplates the use of NOEY2 and activefragments, and NOEY2 nucleic acids, in the screening of compounds foractivity in either stimulating NOEY2 activity, overcoming the lack ofNOEY2, or blocking the effect of a mutant NOEY2 molecule. These assaysmay make use of a variety of different formats and may depend on thekind of “activity” for which the screen is being conducted. Contemplatedfunctional “read-outs” include binding to a compound, inhibition ofbinding to a substrate, ligand, receptor or other binding partner by acompound, phosphatase activity, anti-phosphatase activity,phosphorylation of NOEY2, dephosphorylation of NOEY2, inhibition orstimulation of cell-to-cell signaling, growth, metastasis, celldivision, cell migration, soft agar colony formation, contactinhibition, invasiveness, angiogenesis, apoptosis, tumor progression orother malignant phenotype.

4.13.1 In Vitro Assays

In one embodiment, the invention is to be applied for the screening ofcompounds that bind to the NOEY2 molecule or fragment thereof. Thepolypeptide or fragment may be either free in solution, fixed to asupport, expressed in or on the surface of a cell. Either thepolypeptide or the compound may be labeled, thereby permittingdetermining of binding.

In another embodiment, the assay may measure the inhibition of bindingof NOEY2 to a natural or artificial substrate or binding partner.Competitive binding assays can be performed in which one of the agents(NOEY2, binding partner or compound) is labeled. Usually, thepolypeptide will be the labeled species. One may measure the amount offree label versus bound label to determine binding or inhibition ofbinding.

Another technique for high throughput screening of compounds isdescribed in WO 84/03564. Large numbers of small peptide test compoundsare synthesized on a solid substrate, such as plastic pins or some othersurface. The peptide test compounds are reacted with NOEY2 and washed.Bound polypeptide is detected by various methods.

Purified NOEY2 can be coated directly onto plates for use in theaforementioned drug screening techniques. However, non-neutralizingantibodies to the polypeptide can be used to immobilize the polypeptideto a solid phase. Also, fusion proteins containing a reactive region(preferably a terminal region) may be used to link the NOEY2 activeregion to a solid phase.

Various cell lines containing wild-type or natural or engineeredmutations in NOEY2 can be used to study various functional attributes ofNOEY2 and how a candidate compound affects these attributes. Methods forengineering mutations are described elsewhere in this document, as arenaturally-occurring mutations in NOEY2 that lead to, contribute toand/or otherwise cause malignancy. In such assays, the compound would beformulated appropriately, given its biochemical nature, and contactedwith a target cell. Depending on the assay, culture may be required. Thecell may then be examined by virtue of a number of different physiologicassays. Alternatively, molecular analysis may be performed in which thefunction of NOEY2, or related pathways, may be explored. This mayinvolve assays such as those for protein expression, enzyme function,substrate utilization, phosphorylation states of various moleculesincluding NOEY2, cAMP levels, mRNA expression (including differentialdisplay of whole cell or polyA RNA) and others.

4.13.2 In Vivo Assays

The present invention also encompasses the use of various animal models.Here, the identity seen between human and mouse NOEY2 provides anexcellent opportunity to examine the function of NOEY2 in a whole animalsystem where it is normally expressed. By developing or isolating mutantcells lines that fail to express normal NOEY2, one can generate cancermodels in mice that will be highly predictive of cancers in humans andother mammals. These models may employ the orthotopic or systemicadministration of tumor cells to mimic primary and/or metastaticcancers. Alternatively, one may induce cancers in animals by providingagents known to be responsible for certain events associated withmalignant transformation and/or tumor progression. Finally, transgenicanimals (discussed below) that lack a wild-type NOEY2 may be utilized asmodels for cancer development and treatment.

Treatment of animals with test compounds will involve the administrationof the compound, in an appropriate form, to the animal. Administrationwill be by any route the could be utilized for clinical or non-clinicalpurposes, including but not limited to oral, nasal, buccal, rectal,vaginal or topical. Alternatively, administration may be byintratracheal instillation, bronchial instillation, intradermal,subcutaneous, intramuscular, intraperitoneal or intravenous injection.Specifically contemplated are systemic intravenous injection, regionaladministration via blood or lymph supply and intratumoral injection.

Determining the effectiveness of a compound in vivo may involve avariety of different criteria. Such criteria include, but are notlimited to, survival, reduction of tumor burden or mass, arrest orslowing of tumor progression, elimination of tumors, inhibition orprevention of metastasis, increased activity level, improvement inimmune effector function and improved food intake.

4.13.3 Rational Drug Design

The goal of rational drug design is to produce structural analogs ofbiologically active polypeptides or compounds with which they interact(agonists, antagonists, inhibitors, binding partners, etc.). By creatingsuch analogs, it is possible to fashion drugs which are more active orstable than the natural molecules, which have different susceptibilityto alteration or which may affect the function of various othermolecules. In one approach, one would generate a three-dimensionalstructure for NOEY2 or a fragment thereof. This could be accomplished byx-ray crystallography, computer modeling or by a combination of bothapproaches. An alternative approach, “alanine scan,” involves the randomreplacement of residues throughout molecule with alanine, and theresulting affect on function determined.

It also is possible to isolate a NOEY2-specific antibody, selected by afunctional assay, and then solve its crystal structure. In principle,this approach yields a pharmacore upon which subsequent drug design canbe based. It is possible to bypass protein crystallography altogether bygenerating anti-idiotypic antibodies to a functional, pharmacologicallyactive antibody. As a mirror image of a mirror image, the binding siteof anti-idiotype would be expected to be an analog of the originalantigen. The anti-idiotype could then be used to identify and isolatepeptides from banks of chemically- or biologically-produced peptides.Selected peptides would then serve as the pharmacore. Anti-idiotypes maybe generated using the methods described herein for producingantibodies, using an antibody as the antigen.

Thus, one may design drugs which have improved NOEY2 activity or whichact as stimulators, inhibitors, agonists, antagonists or NOEY2 ormolecules affected by NOEY2 function. By virtue of the availability ofcloned 1p31 sequences described herein, sufficient amounts of NOEY2 canbe produced to perform crystallographic studies. In addition, knowledgeof the polypeptide sequences permits computer employed predictions ofstructure-function relationships.

4.14 Pharmaceutical Compositions and Formulations

Where clinical applications involving NOEY2 compositions arecontemplated, it is often necessary to prepare pharmaceuticalcompositions (comprising e.g., expression vectors, virus stocks,polypeptides, polynucleotides, antibodies and/or drugs) in a formappropriate for the intended application. Generally, this will entailpreparing compositions that are essentially free of pyrogens, as well asother impurities that could be harmful to humans or animals.

One will generally desire to employ appropriate salts and buffers torender delivery vectors stable and allow for uptake by target cells.Buffers also will be employed when recombinant cells are introduced intoa human or a non-human animal. Aqueous compositions of the presentinvention comprise an effective amount of the vector to cells, dissolvedor dispersed in a pharmaceutically acceptable carrier or aqueous medium.Such compositions also are referred to as inocula. The phrase“pharmaceutically or pharmacologically acceptable” refer to molecularentities and compositions that do not produce adverse, allergic, orother untoward reactions when administered to an animal or a human. Asused herein, “pharmaceutically acceptable carrier” includes any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutically active substances is wellknow in the art. Except insofar as any conventional media or agent isincompatible with the vectors or cells of the present invention, its usein therapeutic compositions is contemplated. Supplementary activeingredients also can be incorporated into the compositions.

The active compositions of the present invention may include classicpharmaceutical preparations. Administration of these compositionsaccording to the present invention will be via any common route so longas the target tissue is available via that route. This includes oral,nasal, buccal, rectal, vaginal or topical. Alternatively, administrationmay be by orthotopic, intradermal, subcutaneous, intramuscular,intraperitoneal or intravenous injection. Such compositions wouldnormally be administered as pharmaceutically acceptable compositions,described supra.

The active compounds may also be administered parenterally orintraperitoneally. Solutions of the active compounds as free base orpharmacologically acceptable salts can be prepared in water suitablymixed with a surfactant, such as hydroxypropylcellulose. Dispersions canalso be prepared in glycerol, liquid polyethylene glycols, and mixturesthereof and in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), suitable mixtures thereof,and vegetable oils. The proper fluidity can be maintained, for example,by the use of a coating, such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial an antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutical active substances is well knownin the art. Except insofar as any conventional media or agent isincompatible with the active ingredient, its use in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions.

For oral administration the polypeptides of the present invention may beincorporated with excipients and used in the form of non-ingestiblemouthwashes and dentifrices. A mouthwash may be prepared incorporatingthe active ingredient in the required amount in an appropriate solvent,such as a sodium borate solution (Dobell's Solution). Alternatively, theactive ingredient may be incorporated into an antiseptic wash containingsodium borate, glycerin and potassium bicarbonate. The active ingredientmay also be dispersed in dentifrices, including: gels, pastes, powdersand slurries. The active ingredient may be added in a therapeuticallyeffective amount to a paste dentifrice that may include water, binders,abrasives, flavoring agents, foaming agents, and humectants.

The compositions of the present invention may be formulated in a neutralor salt form. Pharmaceutically-acceptable salts include the acidaddition salts (formed with the free amino groups of the protein) andwhich are formed with inorganic acids such as, for example, hydrochloricor phosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups canalso be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, histidine, procaine and thelike.

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeuticallyeffective. The formulations are easily administered in a variety ofdosage forms such as injectable solutions, drug release capsules and thelike. For parenteral administration in an aqueous solution, for example,the solution should be suitably buffered if necessary and the liquiddiluent first rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, sterile aqueous media which can be employed will be known tothose of skill in the art in light of the present disclosure. Forexample, one dosage could be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion, (see for example, “Remington's PharmaceuticalSciences” 15th Edition, pages 1035–1038 and 1570–1580). Some variationin dosage will necessarily occur depending on the condition of thesubject being treated. The person responsible for administration will,in any event, determine the appropriate dose for the individual subject.Moreover, for human administration, preparations should meet sterility,pyrogenicity, general safety and purity standards as required by FDAOffice of Biologics standards.

4.15 Transgenic Animals

In one embodiment of the invention, a transgenic animal is producedwhich contains one or more functional transgenes that encode afunctional NOEY2 polypeptide or a variant thereof. Transgenic animalsexpressing NOEY2 transgenes, recombinant cell lines derived from suchanimals and transgenic embryos may be useful in methods for screeningfor and identifying agents that induce or repress function of NOEY2.Transgenic animals of the present invention may also be used as modelsfor studying indications such as cancers.

In one embodiment of the invention, a NOEY2 transgene is introduced intoa non-human host to produce a transgenic animal expressing a NOEY2 gene.The transgenic animal is produced by the integration of the transgeneinto the genome in a manner that permits the expression of thetransgene. Methods for producing transgenic animals are generallydescribed by Wagner and Hoppe (U.S. Pat. No. 4,873,191; which isincorporated herein by reference), Brinster et al. 1985; which isincorporated herein by reference in its entirety) and in “Manipulatingthe Mouse Embryo; A Laboratory Manual” 2nd edition (eds., Hogan,Beddington, Costantimi and Long, Cold Spring Harbor Laboratory Press,1994; which is incorporated herein by reference in its entirety).Exemplary non-human animals include mice, rats, monkeys, hamsters, pigs,dogs, cats, goats, rabbits, horses, sheep, or virtually any other animalfor which methods have been developed for introducing a stable transgeneinto its germline.

It may be desirable to replace the endogenous NOEY2 gene(s) byhomologous recombination between the transgene and the endogenous gene;or the endogenous gene may be eliminated by deletion as in thepreparation of “knock-out” animals. Typically, a NOEY2 gene flanked bygenomic sequences is transferred by microinjection into a fertilizedegg. The microinjected eggs are implanted into a host female, and theprogeny are screened for the expression of the transgene. Transgenicanimals may be produced from the fertilized eggs from a number ofanimals including, but not limited to reptiles, amphibians, birds,mammals, and fish. Within a particularly preferred embodiment,transgenic mice are generated which overexpress NOEY2 or express amutant form of the polypeptide. Alternatively, the absence of NOEY2 in“knock-out” mice permits the study of the effects that loss of NOEY2protein has on a cell in vivo. Knock-out mice also provide a model forthe development of NOEY2-related cancers, and particularly ovarian andbreast cancers.

As noted above, transgenic animals and cell lines derived from suchanimals may find use in certain testing studies. In this regard,transgenic animals and cell lines capable of expressing wild-type ormutant NOEY2 may be exposed to test substances. These test substancescan be screened for the ability to enhance wild-type NOEY2 expressionand or function or impair the expression or function of mutant NOEY2.

4.16 Mutagenesis Methods

In certain aspects of the invention, it is desirable to introduce one ormore mutations, insertions, or deletions into a polynucleotide encodinga NOEY2 or NOEY2-like polypeptide. The means for mutagenizing nucleicacid segments are well-known to those of skill in the art. Modificationsto such promoter regions may be made by random, or site-specificmutagenesis procedures. The promoter region may be modified by alteringits structure through the addition or deletion of one or morenucleotides from the sequence which encodes the correspondingun-modified promoter region.

Mutagenesis may be performed in accordance with any of the techniquesknown in the art such as and not limited to synthesizing anoligonucleotide having one or more mutations within the sequence of aparticular promoter region. In particular, site-specific mutagenesis isa technique useful in the preparation of promoter mutants, throughspecific mutagenesis of the underlying DNA. The technique furtherprovides a ready ability to prepare and test sequence variants, forexample, incorporating one or more of the foregoing considerations, byintroducing one or more nucleotide sequence changes into the DNA.Site-specific mutagenesis allows the production of mutants through theuse of specific oligonucleotide sequences which encode the DNA sequenceof the desired mutation, as well as a sufficient number of adjacentnucleotides, to provide a primer sequence of sufficient size andsequence complexity to form a stable duplex on both sides of thedeletion junction being traversed. Typically, a primer of about 17 toabout 75 nucleotides or more in length is preferred, with about 10 toabout 25 or more residues on both sides of the junction of the sequencebeing altered.

The technique of site-specific mutagenesis is well known in the art, asexemplified by various publications. As will be appreciated, thetechnique typically employs a phage vector which exists in both a singlestranded and double stranded form. Typical vectors useful insite-directed mutagenesis include vectors such as the M13 phage. Thesephage are readily commercially available and their use is generally wellknown to those skilled in the art. Double stranded plasmids are alsoroutinely employed in site directed mutagenesis which eliminates thestep of transferring the gene of interest from a plasmid to a phage.

In general, site-directed mutagenesis in accordance herewith isperformed by first obtaining a single-stranded vector or melting apartof two strands of a double stranded vector which includes within itssequence a DNA sequence which encodes the desired promoter region orpeptide. An oligonucleotide primer bearing the desired mutated sequenceis prepared, generally synthetically. This primer is then annealed withthe single-stranded vector, and subjected to DNA polymerizing enzymessuch as E. coli polymerase I Klenow fragment, in order to complete thesynthesis of the mutation-bearing strand. Thus, a heteroduplex is formedwherein one strand encodes the original non-mutated sequence and thesecond strand bears the desired mutation. This heteroduplex vector isthen used to transform or transfect appropriate cells, such as E. colicells, and clones are selected which include recombinant vectors bearingthe mutated sequence arrangement. A genetic selection scheme was devisedby Kunkel et al. (1987) to enrich for clones incorporating the mutagenicoligonucleotide. Alternatively, the use of PCR™ with commerciallyavailable thermostable enzymes such as Taq polymerase may be used toincorporate a mutagenic oligonucleotide primer into an amplified DNAfragment that can then be cloned into an appropriate cloning orexpression vector. The PCR™-mediated mutagenesis procedures of Tomic etal. (1990) and Upender et al. (1995) provide two examples of suchprotocols. A PCR™ employing a thermostable ligase in addition to athermostable polymerase may also be used to incorporate a phosphorylatedmutagenic oligonucleotide into an amplified DNA fragment that may thenbe cloned into an appropriate cloning or expression vector. Themutagenesis procedure described by Michael (1994) provides an example ofone such protocol.

The preparation of sequence variants of the selected promoter-encodingDNA segments using site-directed mutagenesis is provided as a means ofproducing potentially useful species and is not meant to be limiting asthere are other ways in which sequence variants of DNA sequences may beobtained. For example, recombinant vectors encoding the desired promotersequence may be treated with mutagenic agents, such as hydroxylamine, toobtain sequence variants.

As used herein, the term “oligonucleotide directed mutagenesisprocedure” refers to template-dependent processes and vector-mediatedpropagation which result in an increase in the concentration of aspecific nucleic acid molecule relative to its initial concentration, orin an increase in the concentration of a detectable signal, such asamplification. As used herein, the term “oligonucleotide directedmutagenesis procedure” also is intended to refer to a process thatinvolves the template-dependent extension of a primer molecule. The termtemplate-dependent process refers to nucleic acid synthesis of an RNA ora DNA molecule wherein the sequence of the newly synthesized strand ofnucleic acid is dictated by the well-known rules of complementary basepairing (see, for example, Watson, 1987). Typically, vector mediatedmethodologies involve the introduction of the nucleic acid fragment intoa DNA or RNA vector, the clonal amplification of the vector, and therecovery of the amplified nucleic acid fragment. Examples of suchmethodologies are provided by U.S. Pat. No. 4,237,224, specificallyincorporated herein by reference in its entirety.

A number of template dependent processes are available to amplify thetarget sequences of interest present in a sample. One of the best knownamplification methods is the polymerase chain reaction (PCR™) which isdescribed in detail in U.S. Pat. Nos. 4,683,195, 4,683,202 and4,800,159, each of which is incorporated herein by reference in itsentirety. Briefly, in PCR™, two primer sequences are prepared which arecomplementary to regions on opposite complementary strands of the targetsequence. An excess of deoxynucleoside triphosphates are added to areaction mixture along with a DNA polymerase (e.g., Taq polymerase). Ifthe target sequence is present in a sample, the primers will bind to thetarget and the polymerase will cause the primers to be extended alongthe target sequence by adding on nucleotides. By raising and loweringthe temperature of the reaction mixture, the extended primers willdissociate from the target to form reaction products, excess primerswill bind to the target and to the reaction products and the process isrepeated. Preferably a reverse transcriptase PCR™ amplificationprocedure may be performed in order to quantify the amount of mRNAamplified. Polymerase chain reaction methodologies are well known in theart.

Another method for amplification is the ligase chain reaction (referredto as LCR), disclosed in Eur. Pat. Appl. Publ. No. 320,308, incorporatedherein by reference in its entirety. In LCR, two complementary probepairs are prepared, and in the presence of the target sequence, eachpair will bind to opposite complementary strands of the target such thatthey abut. In the presence of a ligase, the two probe pairs will link toform a single unit. By temperature cycling, as in PCR™, bound ligatedunits dissociate from the target and then serve as “target sequences”for ligation of excess probe pairs. U.S. Pat. No. 4,883,750,incorporated herein by reference in its entirety, describes analternative method of amplification similar to LCR for binding probepairs to a target sequence.

Qbeta Replicase, described in PCT Intl. Pat. Appl. Publ. No.PCT/US87/00880, incorporated herein by reference in its entirety, mayalso be used as still another amplification method in the presentinvention. In this method, a replicative sequence of RNA which has aregion complementary to that of a target is added to a sample in thepresence of an RNA polymerase. The polymerase will copy the replicativesequence which can then be detected.

An isothermal amplification method, in which restriction endonucleasesand ligases are used to achieve the amplification of target moleculesthat contain nucleotide 5′-[α-thio]triphosphates in one strand of arestriction site (Walker et al., 1992, incorporated herein by referencein its entirety), may also be useful in the amplification of nucleicacids in the present invention.

Strand Displacement Amplification (SDA) is another method of carryingout isothermal amplification of nucleic acids which involves multiplerounds of strand displacement and synthesis, e.g., nick translation. Asimilar method, called Repair Chain Reaction (RCR) is another method ofamplification which may be useful in the present invention and isinvolves annealing several probes throughout a region targeted foramplification, followed by a repair reaction in which only two of thefour bases are present. The other two bases can be added as biotinylatedderivatives for easy detection. A similar approach is used in SDA.

Still other amplification methods described in Great Britain Pat. Appl.No. 2,202,328, and in PCT Intl. Pat. Appl. Publ. No. PCT/US89/01025,each of which is incorporated herein by reference in its entirety, maybe used in accordance with the present invention. In the formerapplication, “modified” primers are used in a PCR like, template andenzyme dependent synthesis. The primers may be modified by labeling witha capture moiety (e.g., biotin) and/or a detector moiety (e.g., enzyme).In the latter application, an excess of labeled probes are added to asample. In the presence of the target sequence, the probe binds and iscleaved catalytically. After cleavage, the target sequence is releasedintact to be bound by excess probe. Cleavage of the labeled probesignals the presence of the target sequence.

Other nucleic acid amplification procedures include transcription-basedamplification systems (TAS) (Kwoh et al., 1989; PCT Intl. Pat. Appl.Publ. No. WO 88/10315, incorporated herein by reference in itsentirety), including nucleic acid sequence based amplification (NASBA)and 3SR. In NASBA, the nucleic acids can be prepared for amplificationby standard phenol/chloroform extraction, heat denaturation of a sample,treatment with lysis buffer and minispin columns for isolation of DNAand RNA or guanidinium chloride extraction of RNA. These amplificationtechniques involve annealing a primer which has crystal protein-specificsequences. Following polymerization, DNA/RNA hybrids are digested withRNase H while double stranded DNA molecules are heat denatured again. Ineither case the single stranded DNA is made fully double stranded byaddition of second crystal protein-specific primer, followed bypolymerization. The double stranded DNA molecules are then multiplytranscribed by a polymerase such as T7 or SP6. In an isothermal cyclicreaction, the RNAs are reverse transcribed into double stranded DNA, andtranscribed once against with a polymerase such as T7 or SP6. Theresulting products, whether truncated or complete, indicate crystalprotein-specific sequences.

Eur. Pat. Appl. Publ. No. 329,822, incorporated herein by reference inits entirety, disclose a nucleic acid amplification process involvingcyclically synthesizing single-stranded RNA (“ssRNA”), ssDNA, anddouble-stranded DNA (dsDNA), which may be used in accordance with thepresent invention. The ssRNA is a first template for a first primeroligonucleotide, which is elongated by reverse transcriptase(RNA-dependent DNA polymerase). The RNA is then removed from resultingDNA:RNA duplex by the action of ribonuclease H(RNase H, an RNasespecific for RNA in a duplex with either DNA or RNA). The resultantssDNA is a second template for a second primer, which also includes thesequences of an RNA polymerase promoter (exemplified by T7 RNApolymerase) 5′ to its homology to its template. This primer is thenextended by DNA polymerase (exemplified by the large “Klenow” fragmentof E. coli DNA polymerase I), resulting as a double-stranded DNA(“dsDNA”) molecule, having a sequence identical to that of the originalRNA between the primers and having additionally, at one end, a promotersequence. This promoter sequence can be used by the appropriate RNApolymerase to make many RNA copies of the DNA. These copies can thenre-enter the cycle leading to very swift amplification. With properchoice of enzymes, this amplification can be done isothermally withoutaddition of enzymes at each cycle. Because of the cyclical nature ofthis process, the starting sequence can be chosen to be in the form ofeither DNA or RNA.

PCT Intl. Pat. Appl. Publ. No. WO 89/06700, incorporated herein byreference in its entirety, disclose a nucleic acid sequenceamplification scheme based on the hybridization of a promoter/primersequence to a target single-stranded DNA (“ssDNA”) followed bytranscription of many RNA copies of the sequence. This scheme is notcyclic; i.e. new templates are not produced from the resultant RNAtranscripts. Other amplification methods include “RACE” (Frohman, 1990),and “one-sided PCR” (Ohara, 1989) which are well-known to those of skillin the art.

Methods based on ligation of two (or more) oligonucleotides in thepresence of nucleic acid having the sequence of the resulting“di-oligonucleotide”, thereby amplifying the di-oligonucleotide (Wu andDean, 1996, incorporated herein by reference in its entirety), may alsobe used in the amplification of DNA sequences of the present invention.

4.17 Biological Functional Equivalents

In certain embodiments of the invention, one may desire to mutagenize aNOEY2 polypeptide or polynucleotide to make a modification and/or changein the structure of the NOEY2 proteins or DNA segments which encode themand still obtain a functional molecule that encodes a protein or peptidewith desirable characteristics. The following is a discussion based uponchanging the amino acids of a protein to create an equivalent, or evenan improved, second-generation molecule. The amino acid changes may beachieved by changing the codons of the DNA sequence, according to thecodons given in Table 1.

For example, certain amino acids may be substituted for other aminoacids in a protein structure without appreciable loss of interactivebinding capacity with structures such as, for example, antigen-bindingregions of antibodies or binding sites on substrate molecules. Since itis the interactive capacity and nature of a protein that defines thatprotein's biological functional activity, certain amino acid sequencesubstitutions can be made in a protein sequence, and, of course, itsunderlying DNA coding sequence, and nevertheless obtain a protein withlike properties. It is thus contemplated by the inventors that variouschanges may be made in the peptide sequences of the disclosedcompositions, or corresponding DNA sequences which encode said peptideswithout appreciable loss of their biological utility or activity.

In making such changes, the hydropathic index of amino acids may beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte and Doolittle, 1982, incorporate herein byreference). It is accepted that the relative hydropathic character ofthe amino acid contributes to the secondary structure of the resultantprotein, which in turn defines the interaction of the protein with othermolecules, for example, enzymes, substrates, receptors, DNA, antibodies,antigens, and the like.

TABLE 1 Amino Acids Codons Alanine Ala A GCA GCC GCG GCU Cysteine Cys CUGC UGU Aspartic Acid Asp D GAC GAU Glutamic Acid Glu E GAA GAGPhenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGU Histidine HisH CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAA AAG Leucine LeuL UUA UUG CUA CUC CUG CUU Methionine Met M AUG Asparagine Asn N AAC AAUProline Pro P CCA CCC CCG CCU Glutamine Gln Q CAA CAG Arginine Arg R AGAAGG CGA CGC CGG CGU Serine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr TACA ACC ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGGTyrosine Tyr Y UAC UAU

Each amino acid has been assigned a hydropathic index on the basis oftheir hydrophobicity and charge characteristics (Kyte and Doolittle,1982), these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8);phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9);alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8);tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2);glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5);lysine (−3.9); and arginine (−4.5).

It is known in the art that certain amino acids may be substituted byother amino acids having a similar hydropathic index or score and stillresult in a protein with similar biological activity, i.e., still obtaina biological functionally equivalent protein. In making such changes,the substitution of amino acids whose hydropathic indices are within ±2is preferred, those which are within ±1 are particularly preferred, andthose within ±0.5 are even more particularly preferred.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. U.S. Pat.No. 4,554,101, incorporated herein by reference, states that thegreatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with a biologicalproperty of the protein.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4).

It is understood that an amino acid can be substituted for anotherhaving a similar hydrophilicity value and still obtain a biologicallyequivalent, and in particular, an immunologically equivalent protein. Insuch changes, the substitution of amino acids whose hydrophilicityvalues are within ±2 is preferred, those which are within ±1 areparticularly preferred, and those within ±0.5 are even more particularlypreferred.

As outlined above, amino acid substitutions are generally thereforebased on the relative similarity of the amino acid side-chainsubstituents, for example, their hydrophobicity, hydrophilicity, charge,size, and the like. Exemplary substitutions which take various of theforegoing characteristics into consideration are well known to those ofskill in the art and include: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine.

4.18 Methods for Treating NOEY2 Related Malignancies

The present invention also involves, in another embodiment, thetreatment of cancer. The types of cancer that may be treated, accordingto the present invention, is limited only by the involvement of NOEY2.By involvement, it is not even a requirement that NOEY2 be mutated orabnormal—the overexpression of this tumor suppressor may actuallyovercome other lesions within the cell. Thus, it is contemplated that awide variety of tumors may be treated using NOEY2 therapy, particularlythose of the breast and ovaries, but also cancers of the lung, liver,spleen, brain kidney, lymph node, pancreas, small intestine, bloodcells, colon, stomach, endometrium, prostate, testicle, skin, head andneck, esophagus, bone marrow, blood or other tissue.

In many contexts, it is not necessary that the tumor cell be killed orinduced to undergo normal cell death or “apoptosis.” Rather, toaccomplish a meaningful treatment, all that is required is that thetumor growth be slowed to some degree. It may be that the tumor growthis completely blocked, however, or that some tumor regression isachieved. Clinical terminology such as “remission” and “reduction oftumor” burden also are contemplated given their normal usage.

4.18.1 Gene Therapy

One of the therapeutic embodiments contemplated by the present inventorsis the intervention, at the molecular level, in the events involved inthe tumorigenesis of some cancers. Specifically, the present inventorsintend to provide, to a cancer cell, an expression construct capable ofproviding NOEY2 to that cell. Because NOEY2 transcripts have beenidentified not only in humans, but also in rat, mouse, monkey, andhamster, any of these nucleic acids could be used in human or animaltherapy, as could any of the gene sequence variants discussed abovewhich would encode the same, or a biologically equivalent polypeptide.The development and use of such genes for treatment of cancers using a“gene therapy” approach are well known to those of skill in the art.Particularly preferred expression vectors are viral vectors such asadenovirus, adeno-associated virus, herpesvirus, vaccinia virus andretrovirus. Also preferred is an expression vector that is containedwithin, or formulating using encapsulation within a lipid vesicle, lipidparticle, liposome, or liposome-derived composition.

Those of skill in the art are well aware of how to apply gene deliveryto in vivo and ex vivo situations. For viral vectors, one generally willprepare a viral vector stock. Depending on the kind of virus and thetiter attainable, one may deliver anywhere on the order of from about1×10⁴ to about 1×10⁶ infectious particles to the patient. Alternatively,one may deliver higher concentrations of infectious particles to thepatient, on the order of from about 1×10⁹ to about 1×10¹² or higher,depending upon the particular formulation, application, or cancer to betreated. Similar figures may be extrapolated for liposomal or othernon-viral formulations by comparing relative uptake efficiencies.Formulation as a pharmaceutically acceptable composition is known in theart, as discussed supra.

Various routes are contemplated for various tumor types. The sectionbelow on routes contains an extensive list of possible routes. Forpractically any tumor, systemic delivery is contemplated. This willprove especially important for attacking microscopic or metastaticcancer. Where discrete tumor mass may be identified, a variety ofdirect, local and regional approaches may be taken. For example, thetumor may be directly injected with the expression vector. A tumor bedmay be treated prior to, during or after resection. Following resection,one generally will deliver the vector by a catheter left in placefollowing surgery. One may utilize the tumor vasculature to introducethe vector into the tumor by injecting a supporting vein or artery. Amore distal blood supply route also may be utilized.

In a different embodiment, ex vivo gene therapy is contemplated. Thisapproach is particularly suited, although not limited, to treatment ofbone marrow associated cancers. In an ex vivo embodiment, cells from thepatient are removed and maintained outside the body for at least someperiod of time. During this period, a therapy is delivered, after whichthe cells are reintroduced into the patient; hopefully, any tumor cellsin the sample have been killed.

Autologous bone marrow transplant (ABMT) is an example of ex vivo genetherapy. Basically, the notion behind ABMT is that the patient willserve as his or her own bone marrow donor. Thus, a normally lethal doseof irradiation or chemotherapeutic may be delivered to the patient tokill tumor cells, and the bone marrow repopulated with the patients owncells that have been maintained (and perhaps expanded) ex vivo. Because,bone marrow often is contaminated with tumor cells, it is desirable topurge the bone marrow of these cells. Use of gene therapy to accomplishthis goal is yet another way NOEY2 may be utilized according to thepresent invention.

4.18.2 Immunotherapy

Immunotherapeutics, generally, rely on the use of immune effector cellsand molecules to target and destroy cancer cells. The immune effectormay be, for example, an antibody specific for some marker on the surfaceof a tumor cell. The antibody alone may serve as an effector of therapyor it may recruit other cells to actually effect cell killing. Theantibody also may be conjugated to a drug or toxin (chemotherapeutic,radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) andserve merely as a targeting agent. Alternatively, the effector may be alymphocyte carrying a surface molecule that interacts, either directlyor indirectly, with a tumor cell target. Various effector cells includecytotoxic T cells and NK cells.

According to the present invention, it is unlikely that NOEY2 couldserve as a target for an immune effector given that (a) it is unlikelyto be expressed on the surface of the cell and (b) that the presence,not absence, of NOEY2 is associated with the normal state. However, itis possible that particular mutant forms of NOEY2 may be targeted byimmunotherapy, either using antibodies, antibody conjugates or immuneeffector cells.

A more likely scenario is that immunotherapy could be used as part of acombined therapy, in conjunction with NOEY2-targeted gene therapy. Thegeneral approach for combined therapy is discussed below. Generally, thetumor cell must bear some marker that is amenable to targeting, i.e., isnot present on the majority of other cells. Many tumor marker exist andany of these may be suitable for targeting in the context of the presentinvention. Common tumor markers include carcinoembryonic antigen,prostate specific antigen, urinary tumor associated antigen, fetalantigen, tyrosinase (p97), gp68, TAG-72, HMFG, Sialyl Lewis Antigen,MucA, MucB, PLAP, estrogen receptor, laminin receptor, erb B and p155.

4.18.3 Protein Therapy

Another therapy approach is the provision, to a subject, of NOEY2polypeptide, active fragments, synthetic peptides, mimetics or otheranalogs thereof. The protein may be produced by recombinant expressionmeans or, if small enough, generated by an automated peptidesynthesizer. Formulations would be selected based on the route ofadministration and purpose including, but not limited to, liposomalformulations and classic pharmaceutical preparations.

4.18.4 Combined Therapy with Immunotherapy, Chemotherapy or Radiotherapy

Tumor cell resistance to DNA damaging agents represents a major problemin clinical oncology. One goal of current cancer research is to findways to improve the efficacy of chemo- and radiotherapy. One way is bycombining such traditional therapies with gene therapy. For example, theherpes simplex-thymidine kinase (HS-tk) gene, when delivered to braintumors by a retroviral vector system, successfully inducedsusceptibility to the antiviral agent ganciclovir (Culver et al., 1992).In the context of the present invention, it is contemplated that NOEY2replacement therapy could be used similarly in conjunction with chemo-or radiotherapeutic intervention. It also may prove effective to combineNOEY2 gene therapy with immunotherapy, as described above.

To kill cells, inhibit cell growth, inhibit metastasis, inhibitangiogenesis or otherwise reverse or reduce the malignant phenotype oftumor cells, using the methods and compositions of the presentinvention, one would generally contact a “target” cell with a NOEY2expression construct and at least one other agent. These compositionswould be provided in a combined amount effective to kill or inhibitproliferation of the cell. This process may involve contacting the cellswith the expression construct and the agent(s) or factor(s) at the sametime. This may be achieved by contacting the cell with a singlecomposition or pharmacological formulation that includes both agents, orby contacting the cell with two distinct compositions or formulations,at the same time, wherein one composition includes the expressionconstruct and the other includes the agent.

Alternatively, the gene therapy treatment may precede or follow theother agent treatment by intervals ranging from min to wk. Inembodiments where the other agent and expression construct are appliedseparately to the cell, one would generally ensure that a significantperiod of time did not expire between the time of each delivery, suchthat the agent and expression construct would still be able to exert anadvantageously combined effect on the cell. In such instances, it iscontemplated that one would contact the cell with both modalities withinabout 12–24 h of each other and, more preferably, within about 6–12 h ofeach other, with a delay time of only about 12 h being most preferred.In some situations, it may be desirable to extend the time period fortreatment significantly, however, where several days (2, 3, 4, 5, 6 or7) to several wk (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respectiveadministrations.

It also is conceivable that more than one administration of either NOEY2or the other agent will be desired. Various combinations may beemployed, where NOEY2 is “A” and the other agent is “B”, as exemplifiedbelow:

A/B/A B/A/B B/B/A A/A/B B/A/A A/B/B B/B/B/A B/B/A/B

A/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B B/B/B/A

A/A/A/B B/A/A/A A/B/A/A A/A/B/A A/B/B/B B/A/B/B B/B/A/B

Other combinations are contemplated. Again, to achieve cell killing,both agents are delivered to a cell in a combined amount effective tokill the cell.

Agents or factors suitable for use in a combined therapy are anychemical compound or treatment method that induces DNA damage whenapplied to a cell. Such agents and factors include radiation and wavesthat induce DNA damage such as, γ-irradiation, X-rays, UV-irradiation,microwaves, electronic emissions, and the like. A variety of chemicalcompounds, also described as “chemotherapeutic agents,” function toinduce DNA damage, all of which are intended to be of use in thecombined treatment methods disclosed herein. Chemotherapeutic agentscontemplated to be of use, include, e.g., adriamycin, 5-fluorouracil(5FU), etoposide (VP-16), camptothecin, actinomycin-D, mitomycin C,cisplatin (CDDP) and even hydrogen peroxide. The invention alsoencompasses the use of a combination of one or more DNA damaging agents,whether radiation-based or actual compounds, such as the use of X-rayswith cisplatin or the use of cisplatin with etoposide. In certainembodiments, the use of cisplatin in combination with a NOEY2 expressionconstruct is particularly preferred as this compound.

In treating cancer according to the invention, one would contact thetumor cells with an agent in addition to the expression construct. Thismay be achieved by irradiating the localized tumor site with radiationsuch as X-rays, UV-light, γ-rays or even microwaves. Alternatively, thetumor cells may be contacted with the agent by administering to thesubject a therapeutically effective amount of a pharmaceuticalcomposition comprising a compound such as, adriamycin, 5-fluorouracil,etoposide, camptothecin, actinomycin-D, mitomycin C, or more preferably,cisplatin. The agent may be prepared and used as a combined therapeuticcomposition, or kit, by combining it with a NOEY2 expression construct,as described above.

Agents that directly cross-link nucleic acids, specifically DNA, areenvisaged to facilitate DNA damage leading to a synergistic,antineoplastic combination with NOEY2. Agents such as cisplatin, andother DNA alkylating agents may be used. Cisplatin has been widely usedto treat cancer, with efficacious doses used in clinical applications of20 mg/m² for 5 days every three wk for a total of three courses.Cisplatin is not absorbed orally and must therefore be delivered viainjection intravenously, subcutaneously, intratumorally orintraperitoneally.

Agents that damage DNA also include compounds that interfere with DNAreplication, mitosis and chromosomal segregation. Such chemotherapeuticcompounds include adriamycin, also known as doxorubicin, etoposide,verapamil, podophyllotoxin, and the like. Widely used in a clinicalsetting for the treatment of neoplasms, these compounds are administeredthrough bolus injections intravenously at doses ranging from 25–75 mg/mat 21 day intervals for adriamycin, to 35–50 mg/m² for etoposideintravenously or double the intravenous dose orally.

Agents that disrupt the synthesis and fidelity of nucleic acidprecursors and subunits also lead to DNA damage. As such a number ofnucleic acid precursors have been developed. Particularly useful areagents that have undergone extensive testing and are readily available.As such, agents such as 5-fluorouracil (5-FU), are preferentially usedby neoplastic tissue, making this agent particularly useful fortargeting to neoplastic cells. Although quite toxic, 5-FU, is applicablein a wide range of carriers, including topical, however intravenousadministration with doses ranging from 3 to 15 mg/kg/day being commonlyused.

Other factors that cause DNA damage and have been used extensivelyinclude what are commonly known as γ-rays, X-rays, and/or the directeddelivery of radioisotopes to tumor cells. Other forms of DNA damagingfactors are also contemplated such as microwaves and UV-irradiation. Itis most likely that all of these factors effect a broad range of damageDNA, on the precursors of DNA, the replication and repair of DNA, andthe assembly and maintenance of chromosomes. Dosage ranges for X-raysrange from daily doses of 50 to 200 roentgens for prolonged periods oftime (3 to 4 wk), to single doses of 2000 to 6000 roentgens. Dosageranges for radioisotopes vary widely, and depend on the half-life of theisotope, the strength and type of radiation emitted, and the uptake bythe neoplastic cells.

The skilled artisan is directed to “Remington's Pharmaceutical Sciences”15th Edition, chapter 33, in particular pages 624–652. Some variation indosage will necessarily occur depending on the condition of the subjectbeing treated. The person responsible for administration will, in anyevent, determine the appropriate dose for the individual subject.Moreover, for human administration, preparations should meet sterility,pyrogenicity, general safety and purity standards as required by FDAOffice of Biologics standards.

The inventors propose that the regional delivery of NOEY2 expressionconstructs to patients with NOEY2-linked cancers will be a veryefficient method for delivering a therapeutically effective gene tocounteract the clinical disease, and particularly to cancers such asovarian and breast cancer. Similarly, the chemo- or radiotherapy may bedirected to a particular, affected region of the subjects body.Alternatively, systemic delivery of expression construct and/or theagent may be appropriate in certain circumstances, for example, whereextensive metastasis has occurred.

In addition to combining NOEY2-targeted therapies with chemo- andradiotherapies, it also is contemplated that combination with other genetherapies will be advantageous. For example, targeting of NOEY2 and p53or p16 mutations at the same time may produce an improved anti-cancertreatment. Any other tumor-related gene conceivably can be targeted inthis manner, for example, p21, Rb, APC, DCC, NF-1, NF-2, BCRA2, p16,FHIT, WT-1, MEN-I, MEN-II, BRCA1, VHL, FCC, MCC, ras, myc, neu, raf erb,src, fms, jun, trk, ret, gsp, hst, bcl and abl.

It also should be pointed out that any of the foregoing therapies mayprove useful by themselves in treating a NOEY2-related cancer. In thisregard, reference to chemotherapeutics and non-NOEY2 gene therapy incombination should also be read as a contemplation that these approachesmay be employed separately.

4.19 ELISAs and Immunoprecipitation

ELISAs may be used in conjunction with the invention. In an ELISA assay,proteins or peptides incorporating tumor suppressor protein antigensequences are immobilized onto a selected surface, preferably a surfaceexhibiting a protein affinity such as the wells of a polystyrenemicrotiter plate. After washing to remove incompletely adsorbedmaterial, it is desirable to bind or coat the assay plate wells with anonspecific protein that is known to be antigenically neutral withregard to the test antisera such as bovine serum albumin (BSA), caseinor solutions of milk powder. This allows for blocking of nonspecificadsorption sites on the immobilizing surface and thus reduces thebackground caused by nonspecific binding of antisera onto the surface.

After binding of antigenic material to the well, coating with anon-reactive material to reduce background, and washing to removeunbound material, the immobilizing surface is contacted with theantisera or clinical or biological extract to be tested in a mannerconducive to immune complex (antigen/antibody) formation. Suchconditions preferably include diluting the antisera with diluents suchas BSA, bovine gamma globulin (BGG) and phosphate buffered saline(PBS)/Tween®. These added agents also tend to assist in the reduction ofnonspecific background. The layered antisera is then allowed to incubatefor from about 2 to about 4 h, at temperatures preferably on the orderof about 25° to about 27° C. Following incubation, theantisera-contacted surface is washed so as to remove non-immunocomplexedmaterial. A preferred washing procedure includes washing with a solutionsuch as PBS/Tween®, or borate buffer.

Following formation of specific immunocomplexes between the test sampleand the bound antigen, and subsequent washing, the occurrence and evenamount of immunocomplex formation may be determined by subjecting sameto a second antibody having specificity for the first. To provide adetecting means, the second antibody will preferably have an associatedenzyme that will generate a color development upon incubating with anappropriate chromogenic substrate. Thus, for example, one will desire tocontact and incubate the antisera-bound surface with a urease orperoxidase-conjugated anti-human IgG for a period of time and underconditions which favor the development of immunocomplex formation (e.g.,incubation for 2 h at room temperature in a PBS-containing solution suchas PBS Tween®).

After incubation with the second enzyme-tagged antibody, and subsequentto washing to remove unbound material, the amount of label is quantifiedby incubation with a chromogenic substrate such as urea and bromocresolpurple or 2,2′-azino-di-(3-ethyl-benzthiazoline)-6-sulfonic acid (ABTS)and H₂O₂, in the case of peroxidase as the enzyme label. Quantitation isthen achieved by measuring the degree of color generation, e.g., using avisible spectra spectrophotometer.

The anti-tumor suppressor protein antibodies of the present inventionare particularly useful for the isolation of other tumor suppressorprotein antigens by immunoprecipitation. Immunoprecipitation involvesthe separation of the target antigen component from a complex mixture,and is used to discriminate or isolate minute amounts of protein. Forthe isolation of membrane proteins cells must be solubilized intodetergent micelles. Nonionic salts are preferred, since other agentssuch as bile salts, precipitate at acid pH or in the presence ofbivalent cations.

In an alternative embodiment the antibodies of the present invention areuseful for the close juxtaposition of two antigens. This is particularlyuseful for increasing the localized concentration of antigens, e.g.enzyme-substrate pairs.

4.20 Western Blots

The NOEY2 compositions of the present invention will find great use inimmunoblot or western blot analysis. The anti-NOEY2 antibodies may beused as high-affinity primary reagents for the identification ofproteins immobilized onto a solid support matrix, such asnitrocellulose, nylon or combinations thereof. In conjunction withimmuno-precipitation, followed by gel electrophoresis, these may be usedas a single step reagent for use in detecting antigens against whichsecondary reagents used in the detection of the antigen cause an adversebackground. This is especially useful when the antigens studied areimmunoglobulins (precluding the use of immunoglobulins binding bacterialcell wall components), the antigens studied cross-react with thedetecting agent, or they migrate at the same relative molecular weightas a cross-reacting signal.

Immunologically-based detection methods for use in conjunction withWestern blotting include enzymatically-, radiolabel-, orfluorescently-tagged secondary antibodies against the toxin moiety areconsidered to be of particular use in this regard.

4.21 Definitions

The following words and phrases have the meanings set forth below:

Expression: The combination of intracellular processes, includingtranscription and translation undergone by a coding DNA molecule such asa structural gene to produce a polypeptide.

Promoter: A recognition site on a DNA sequence or group of DNA sequencesthat provide an expression control element for a structural gene and towhich RNA polymerase specifically binds and initiates RNA synthesis(transcription) of that gene.

Structural gene: A gene that is expressed to produce a polypeptide.

Transformation: A process of introducing an exogenous DNA sequence(e.g., a vector, a recombinant DNA molecule) into a cell or protoplastin which that exogenous DNA is incorporated into a chromosome or iscapable of autonomous replication.

Transformed cell: A cell whose DNA has been altered by the introductionof an exogenous DNA molecule into that cell.

Transgenic cell: Any cell derived or regenerated from a transformed cellor derived from a transgenic cell.

Vector: A DNA molecule capable of replication in a host cell and/or towhich another DNA segment can be operatively linked so as to bring aboutreplication of the attached segment. A plasmid is an exemplary vector.

5.0 EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

5.1 Example 1 Isolation and Characterization of the NOEY2 Gene

The inventors have utilized differential display of mRNA by means of thepolymerase chain reaction (DDPCR™) (Liang and Pardee, 1992) to isolate anovel gene-NOEY2 from normal ovarian epithelial cells, it may serve as atumor suppresser gene in ovarian and breast cancer.

Since 1990, the inventors have adapted a system developed by Dr. NellieAuersperg (Pizon et al., 1988) for maintaining normal human ovariansurface epithelial (OSE) cells in culture to facilitate comparison withmalignant ovarian cancer cell lines or with tumor cells isolateddirectly from ascites fluid. The normal OSE cells culture are obtainedby gently scraping the surface of ovaries from patients undergoingsurgery for nonmalignant gynecological diseases. OSE cells can only bemaintained in culture for 8 to 10 passages. The cell cycle studiesshowed that most of OSE cells stably arrest growth with a GI DNAcontent, only small part of OSE cells had the ability to enter S phase.Since epithelial cystoadenocarcinoma constitute the large majority ofovarian malignancies, OSE cells provide a good model to investigate thiscancer.

Using Differential Display Polymerase Chain Reaction (DDPCR™), theinventors have isolated and cloned a novel gene—NOEY2—it is expressed innormal ovarian epithelial cells but consistently absent ordown-regulated in the ovarian cancer cell lines.

Using rapid amplification of cDNA ends (RACE), the sequence of NOEY2 wasamplified from OSE mRNA template between a defined internal site andunknown 5′ end of the mRNA. The first strand cDNA is synthesized fromovarian epithelial cell RNA using an NOEY2-specific primer SP1, AMVreverse transcriptase and the deoxynucleotide mixture. Then terminaltransferase is used to add a homopolymeric A-tail to the 3′ end of thecDNA. Tailed cDNA is then amplified by PCR™ using a NOEY2 specificprimer SP2 and the oligo dT-anchor primer. As a result the 5′ RACEproducts were cloned into the TA vector for subsequent characterization,which included sequencing and restriction mapping. A cDNA library fromOSE cells constructed in Lambda ZAPII® were screened with the extendedNOEY2 cDNA. 5 μg of mRNA was purified from OSE cells. The ZAP-cDNAsynthesis kit (Stratagene, La Jolla, Calif.) was used to construct OSEcDNA library. This cDNA library provided a method by which thetranscription and processing of ovarian epithelial cell mRNA can beexamined. The extended NOEY2 cDNA from RACE (Boehringer Mannheim,Indianapolis, Ind.) was ³²P-dCTP labeled by a random primer and used forscreening OSE cDNA library. This screening procedure is performed onbacteriophage plaques, the OSE cDNA library is spread out on agaroseplates, then the clones were transferred to filter membranes. The cloneswere hybridized to NOEY2 DNA probe. The positive clones weresubsequently analyzed, they encoded the mRNA sequence and allowedprediction of the amino acid sequence. Using these two techniques, closeto a full-length NOEY2 cDNA sequence and open reading from (ORF) wasobtained (FIG. 1A and FIG. 1B). The nucleotide sequence (SEQ ID NO:1)contains a 5′ untranslated region of 149 bp, an open reading frame of684 bp encoding a protein of 228 amino acids (SEQ ID NO:2) ending with aTGA codon and 646 bp of 3′ untranslated sequence. Translation of theNOEY2 protein yielded a protein of approximately 26 kDa. The nucleotideand amino acid sequence of the ORF was used to search the Genbank ESTdatabase using Netscape search GenBank of National Center forBiotechnology Information (NCBI) in EST database level I and level II.The most significant similarity detected was with members of the Ras andRap gene family. NOEY2 shares 58% amino acid homology with Rap1A, 56%with Rap1B, 58% with Rap 2, 61% with Rap2B, 51% with c-K-Ras and 54%with H-Ras. The NOEY2 gene ORF exhibits three features similar to thoseof Ras/Rap family members: (a) a highly conserved GTP binding domain,(b) a putative effector domain YLPTIENTY (SEQ ID NO:3), and (c) themembrane localizing CAAX motif (where C is cysteine, A is an aliphaticamino acid and X is any amino acid) at the COOH terminus (FIG. 2).

Within the effector domain, however, NOEY2 differs both from Ras and Rapfamily members where the sequence YDPTIEDSY (SEQ ID NO:4) is found inall Ras and Rap genes. NOEY2 instead has YLPTIENTY. NOEY2 sequence hadsubstitutions of alanine for glycine at amino acid 12 and valine forglutamine at amino acid 61 when compared to p21^(ras), consistent withconstitutive activation of the G protein if it behaved in a mannersimilar to Ras.

NOEY2 cDNA was hybridized and analyzed on Northern blots. Total RNA wasisolated form confluent cell cultures using Trizol reagent (GIBCO BRL,Grand Island, N.Y.). RNA samples (15 μg) were size-fractionated byformaldehyde/agarose gel electrophoresis and transferred to ahybond-nylon membrane. The membranes were UV-cross liked, prehybridizedand hybridized to ³²P-labeled NOEY2 DNA probes. Hybridization wasperformed at 42° C. in 10× Denhard's solution and 50% formamide. 1×SSC,10 mM EDTA, 0.1% SDS and 300 μg/ml denatured Salmon Sperm DNA. A NOEY2message of 1.9 kb was expressed in all eleven primary normal OSEcultures and all ten primary normal breast epithelial cultures, but waslost in 11 of 12 ovarian cancer cell lines (FIG. 3A) and 9 of 9 breastcancer cell lines (FIG. 3B). One ovarian cancer cell line CAOV3expressed NOE. Even in this line, however, NOEY2 was detected at lowerlevels than in normal epithelial cells. NOEY2 expression was also lostin each of 9 primary ovarian cancer cell preparations that wereseparated and purified from patients' ascites (FIG. 3C). Using multipletissue blots containing polyA RNA from sixteen different normal humantissues, the NOEY2 gene was found in several other normal tissues,including heart, liver, pancreas, brain, but the highest expressionoccurred in normal ovary (FIG. 3D). The blots were obtained fromClontech (CLONTECH Laboratories, Palo Alto, Calif.), and Northern blotsperformed as described above. The inventors constructed the recombinantplasmids that express GST fusion proteins. The NOEY2 cDNA fragment whichincluded all four GTP binding domains has been obtained by PCR™amplification and fused in-frame into pGEX-2T vectors to producerecombinant constructs GST-NOEY2 which expressed a fusion protein withM_(r) 53,000 (53 kDa), of which 27 kDa was GST and 26 kDa was NOEY2derived. Large quantities of fusion protein were prepared and purifiedby preparative SDS-polyacrylamide gel electrophoresis (PAGE). Rabbitantiserum and monoclonal antibodies are being prepared by a standardprotocol. Eight monoclonal antibodies against GST-NOEY2 have beenobtained and used for detecting NOEY2 protein. By Western blot analysis,a 26 kDa protein were expressed by normal ovarian and breast epithelialcells, but could not be detected in ovarian and breast cancer cell lines(FIG. 3E).

Genes with the ability to regulate growth often have conservedcounterparts in phylogenetically related organisms. The inventorsexamined genomic DNA from four other vertebrate species (mouse NIH3T3,Rat1A, Monkey COS7 and chicken CHO cell lines) by hybridizing with theNOEY2 probe. Under standard hybridization stringencies, homologoussequences were detected in all samples. Genomic DNA was isolated fromconfluent cell cultures by DNAzol reagent (GIBCO BRL Grand Island,N.Y.). DNA samples (50 μg) were digested with restriction enzymes,separated by agarose gel electrophoresis and transferred to ahybond-nylon membrane. The Southern blot hybridization procedure wassame as in Northern blot.

P1 clones from the DuPont P1 Genomic Library were screened with NOEY2specific primer pairs Y2F3/B1-2 or Y2-19T/3Y2SP2 and used to determinetheir locations on human chromosome by fluorescence in situhybridization (FISH). Probe DNA was extracted and labeled withdigoxigenin-11-dUTP by nick translation. The hybridized signal wasdetected by anti-digoxigenin conjugated with FITC. The location of theprobes was determined by digital image microscopy following FISH andlocalized by the fractional length from the p-terminus (Flpter). NOEY2has been mapped to chromosome 1p31 and Bacs obtained. LOH at 1p31 hasbeen observed in a significant percent of breast and ovarian cancer(Kitayama et al., 1989). Using an intragenic TA dinucleotide repeatlength polymorphism as a marker, the inventors have detected LOH withinthe NOEY2 gene in 9 of 18 ovarian cancers (50%).

Sense and antisense constructs of NOEY2 were transfected withlipofectamine into three ovarian cancer cell lines (OVCA433, OVCA429,Hey), one breast cancer cell line (SKBr3) that did not express NOEY2 andone lung cancer cell line that did express NOEY2 (A-549) (FIG. 4A).NOEY2 cDNA was excised from the EcoRI cloning sites from theBluescript/λZapII® vector. This fragment was 1 Kb in size and includedthe ORF, it was inserted into the pcDNA3 neo eukaryotic expressionvector (Invitrogen, San Diego, Calif.) in the sense and antisenseorientation. The constructs were transfected into three ovarian cancercell lines (OVCA433, OVCA429, Hey), one breast cancer cell lines (SKBr3)and one lung cancer cell line (A549) by lipofectamin method (GIBCO BRLGrand Island, N.Y.). As controls, similar amounts of carrier DNA and thevector DNA only were also transfected into cells. The mixtures oflipofectamin and constructs were exposed to the cells for six h at 37°C., then replaced by cell culture medium. After incubation for 48 h at37° C., transfected cells were trypsinized and seeded into 100 mmdishes, select medium with G418 400 μg/ml to 1000 μg/ml was added to thecells. Replaced the select medium every four days. After 2–3 wk,transformed colonies began to appear, the dishes were stained by 0.1%coomassie blue in 30% methanol and 10% acetic acid.

To explore mechanisms underlying growth inhibition, the inventors havedetermined the effect of NOEY2 expression on other growth regulatorymolecules. NOEY2 sense constructs, but not antisense constructs,strongly inhibited cyclin D1 promoter activity when cotransfected with aplasmid containing the luciferase gene under the control of the cyclinD1 promoter in Saos-2 and NIH3T3 as well as ovarian and breast cancercell lines whose growth could be inhibited (FIG. 4B). As cyclin D1 isrequired for G1-S progression (Albanese et al., 1995), the potentinhibition of the cyclin D1 promoter activity by NOEY2 could contributeto the observed growth inhibition. Cyclin D1 expression is upregulatedin 25–30% of ovarian cancers in the absence of gene amplification(Worsley et al., 1997).

Multiple growth factors, including epidermal growth factor (EGF),insulin, hydrocortisone and bovine pituitary extract (BPE) can stimulategrowth of primary breast (FIG. 5A) and ovarian epithelial cells.Increased growth rates were associated with down-regulation of bothNOEY2 and p21^(WAF1/CIP1) in Northern analysis (FIG. 5B). To examine theinteraction between NOEY2 and p21^(WAF1/CIP1), an NOEY2 fragment thatincluded the entire ORF was fused in frame with a plasmid that containeda triple hemagglutinin (HA) repeat and this HA-NOEY2 construct wastransfected into NIH3T3 cells, two color immunofluorscent stainingdemonstrated that HA-NOEY2 transfectants had higher p21^(WAF1/CIP1)protein expression than did nontransfected cells or cells transfectedwith a HA-Erk2 cDNA (FIG. 6A, FIG. 6B, FIG. 6C, and FIG. 6D). Thep21^(WAF1/CIP1) protein has been shown to arrest cell growth byinhibition of cyclin dependent kinases (Xiong et al., 1993). Whilep21^(WAF1/CIP1) is a p53 inducible gene (E1-Deiry et al., 1993),induction of this inhibitor has also been observed in the absence offunctional p53 (Michieli et al., 1994). Similarly, transfections ofNOEY2 into the Hey (p53 wild type), SKBr3 (p53 mutant) and Saos-2 (p53null) cells exerted p53 independent inhibition of cyclin D1 promoteractivity. These observations collectively suggest that NOEY2 functionsas a negative regulator of cell growth probably through interaction withcomponents of cell cycle control.

The genomic sequence of NOEY2 contains two exons and one intron. Theinventors have sequenced the hole coding region and 1.9 Kb upstream toidentify putative mutations in NOEY2 using DNA from different celllines. Eight of the 18 tumor cell lines (SKOv3, OVCAR-3, OVCA429,OVCA432, BT20, MCF-7, MDA-MB-432 and SKBr3) have a heterozygous A to Gsubstitution at −750 (44%), which is also presenting 5 of 36 primaryovarian cancer DNA samples (14%) and 20 of 100 DNA samples from normalperipheral leukocytes (20%). This sequence variation appears to be afunctionally significant polymorphism in that induced mutation of A to Gat −750 reduces the activity of the NOEY2 promoter by more than 50% inOSE and NBE cells (FIG. 8). IN addition, sequence abnormalities havebeen found in the promote region of NDA-MB-468 with C(−13)G in oneallele and G(+99)A in the other. CAOv3 and BT20 each have aG(coding-231)A alteration encoding an Ala to Thr change, which also wasfound in 6 of 110 normal control DNA samples (5%). Only one allele ofNOEY2 was expressed in each of these cell lines as assessed by RT-PCR™cDNA sequence analysis and by digestion with HhaI suggesting acquiredmethylation or germ line imprinting. Hypermethylation of CpG islandssurrounding the TATA box was found in 2 or 8 breast cancer cell lines(MDA-MB4-35 and MDA-MB-453). Three OSE, 2 NBE DNA, 9 ovarian cancer and4 breast cancer cell lines exhibited partial methylation at this site.Expression of only one NOEY2 allele may be important, given the highrate of LOH and the frequent occurrence of a functionally significantpolymorphism at −750 in the promoter region. Considering that lack ofNOEY2 expression has been observed in more than 90% of ovarian andbreast cancer cell lines and freshly isolated ovarian cancer cells,multiple mechanisms, including mutation, hypermethylation andtranscriptional regulation may be important in the loss of NOEY2expression in tumors. The ability of NOEY2 to inhibit tumor growthcombined with LOH and genetic alterations suggest that NOEY2 functionsas a tumor suppressor.

5.2 Example 2 Significance of NOEY2 Expression Loss in Ovarian Cancers

NOEY2 expression may be assessed at the level of message (with probesand primers already available) and protein (with antibodies developed asdescribed below). Expression may be correlated with histology, stage,grade, outcome and response to chemotherapeutic agents. DNA, RNA andprotein may be obtained from primary ovarian epithelial cell cultures,ovarian cancer cell lines, primary ovarian cancer cells from patients'ascites and cryopreserved normal ovary and tumor tissues. Ascites tumorcells may be purified to >95% homogeneity using techniques developed inthe inventors' laboratory (Hurteau et al., 1994).

5.2.1 Materials and Methods

5.2.1.1 Northern Blot Analysis

NOEY2 cDNA probe was labeled with P³²-dCTP by random primer. Fifteenmicrograms of total cellular RNA was separated in 1.2%formaldehyde-agarose gels and immobilized on a Hybond-N membrane bystandard capillary transfer and UV crosslinking, and then prehybridizedand hybridized to NOEY2 probe in 50% formamide. 1×SSC. 10× Denhardt'ssolution, 10 mM EDTA, 0.1% SDS and 300 μg/ml denatured salmon sperm DNAat 42° C. for 24 h. The blot was washed at 42° C. in 0.1×SSC 0.1% SDSbefore exposure. Hybridization of the same blot to a probe for 18S-rRNAindicates an equal amount of RNA in all lanes.

5.2.1.2 Cell Culture of Primary OSE Cells and Ovarian Cancer Cell Lines

Cultures of primary normal OSE cells were obtained by gently scrapingthe surface of ovaries from patients undergoing surgery for nonmaligantgynecological diseases. Epithelial cells were cultured in OSE medium(MCDB105/199 medium supplemented with 15% fetal calf serum and 10 ng/mlEGF). Ovarian cancer cell lines were maintained as previously described(Xu et al., 1991).

5.2.1.3 Purification of Ovarian Cancer Cells from Patient's Ascites

Cryopreserved ovarian cancer ascites tumor cells were thawed andpurified to >95% homogeneity using discontinuous Percoll densitygradient centrifugation and immunoaffinity removal of CD45 positiveinflammatory cells as previously described (Hurteau et al., 1994).

5.2.1.4 Transfection and Colony Formation Assay

NOEY2 cDNA (1 Kb) was released from the EcoRI cloning site in theBluescript/Lambda ZapII® vector and inserted into the pcDNA3-neoeukaryotic expression vector (Invitrogen, Carlsbad, Calif.) in sense andantisense orientations. The constructs were transfected into threeovarian cancer cell lines (OVCA433, OVCA429, Hey), one breast cancercell line (SKBr3) and one lung cancer cell line (A-549) usinglipofectamine. After incubation for 48 h at 37° C., transfected cellswere trypsinized and seeded into 100 mm dishes. Selection medium withG418 (400 μg/ml to 1000 μg/ml) was added. Two wk later, colonies werestained by 0.1% Coomassie blue in 30% methanol and 10% acetic acid.

5.2.1.5 Two Color Immunofluorescent Staining

The HA-NOEY2 and HA-Erk2 constructs were transfected into NIH3T3 cellsusing lipofectamine. After 24 h, cells were fixed with 4%paraformadehyde permeabilized with 0.5% Triton X-100 and stained with ananti-p21^(WAF1/CIP1) polyclonal antibody (c-19) (Santa Cruz) andanti-rabbit IgG FITC conjugate followed by subsequent incubation with ananti-HA-rhodamine conjugate (Boehringer Mannheim, Indianapolis, Ind.).

5.2.1.6 Genbank Accession Number

NOEY2 has the GenBank accession number U96750.

5.2.1.7 Antibody Preparation and Purification

To facilitate studies of protein expression, specific antibodies may beprepared against recombinantly derived NOEY2 fusion proteins. Theinventors have already constructed three recombinant plasmids thatexpress GST fusion proteins. Three fragments (of 0.8 Kb, 0.6 Kb and 0.4Kb) have been obtained from NOEY2 cDNA clones by PCR™ amplification. Oneincluded all four GTP binding domains, the second included the third andfourth domains and the third included the first and second domains. TheGST-NOEY2 fusion proteins were prepared and purified by preparativeSDS-polyacrylamide gel electrophoresis (PAGE). Rabbits have beenimmunized with the NOEY2 fusion proteins and initial bleeds may beavailable in the next few wk. This approach has been utilized togenerate antibodies against a number of proteins including signalingmolecules (Gibson et al., 1993). To enrich for antibodies recognizingonly NOEY2 protein determinants, two affinity columns may be prepared,one with GST protein and the other with the fusion protein. Antisera maybe passed through the fusion protein/sepharose column and eluted withglycine buffer (pH 2.3). The eluate may be neutralized and passedthrough the GST column several times to remove antibody directed againstGST.

Anti-NOEY2 murine monoclonal antibodies may be prepared and screenedusing standard protocols which the inventors have previously used indevelopment of OC125 (Bast et al., 1981) which binds CA125 antigen (Bastet al., 1983). Hybridomas may be screened against the immunizingGST-fusion protein and against GST by ELISA to identify those whichspecifically react with NOEY2. Polyclonal and monoclonal antibodies maybe assessed for utility in immuno-precipitation, western blotting,immunohistochemistry and immunofluorescence utilizing the immunizingfusion protein as control.

5.2.1.8 Statistical Analysis

Patient outcome may be characterized by one of the time-to-eventvariables 1) survival time or 2) disease-free survival (DFS) or by thebinary variable indicating either 3) response or 4) partial response tochemotherapy (Cox, 1972; Modern Applied Statistics with S-Plus, 1994;Grambsch and Therneau, 1994; Harrington and Fleming, 1991). Each ofthese evaluations may be carried out by regression analysis, with thepatient outcome as the response variable in the regression model andNOEY2 included as a predictive covariate along with the establishedpredictors disease stage, disease grade, amount of residual disease postsurgery, and other molecular markers, including HER-2, EGFR, fms, andp53. Because NOEY2 is recorded as an ordinal variable taking on thevalues 0,1,2,3 or 4, it may be evaluated first as a numerical covariateand subsequently as a categorical covariate in each regression analysis.For each patient outcome, specific questions to be addressed includewhether NOEY2 per se is predictive, if so whether the effect of NOEY2 onpatient outcomes survival of DFS changes over time, and whether anysignificant effect of NOEY2 persists in the presence of the establishedpredictors noted above. Relationships between pairs of covariates may beevaluated by computing standard Pearson correlations and Spearman rankcorrelations between numerical variables and constructing their smoothedscattergrams, by cross-tabulating categorical variables, and by carryingout Kruskal-Wallis or Wilcoxon-Mann-Whitney tests to assess the changeof a numerical variable across a categorical variable.

Covariate effects on each of the time-to-event outcomes survival and DFSmay be evaluated using the Cox proportional hazards regression model(Cox, 1972) and its extensions (Harrington and Fleming, 1991; Therneau,1994). Goodness-of-fit may be evaluated using the methods of Therneau etal., (1990) and Lin (1991). Smoothed martingale residual plots may beused to determine if any of these markers have a possible “thresholdeffect” on survival or DFS (Kornblau et al., 1995; Hilsenbeck and Clark,1996). Possible time-varying covariate effects may be identified andevaluated using the method of Grambsch and Therneau (Grambsch andTherneau, 1994). The amount of variation in survival or DFS explained byNOEY2 and the other covariates may be quantified using the methods ofKorn and Simon (1990) and Schemper and Stare (1996). Statisticalsoftware for fitting these models has been obtained from T. Therneau(1994).

Effects of NOEY2 and the other covariates on the binary patient outcomesresponse or partial response may be evaluated using binomial regression,including logistic regression and probit analysis as special cases.Goodness-of-fit may be evaluated using the methods of Pregibon (1982).

5.2.1.9 Sequencing and Methylation

After SSCP analysis of the coding region. PCR™ fragments with abnormalmobility were cloned into the PCR-Script Amp SK (+) cloning vector(Stratagene, LaJolla, Calif.). More than 10 clones of each sample weresequenced using an ABI PRISM 377 DNA automatic sequencer and a Big Dye™terminator cycle sequencing kit. Upstream promoter region sequences (1.9Kb) were amplified by four pairs of primers with a −21M13 tail. PurifiedPCR™ products were directly sequenced using −21M13 Dye™ primer cyclesequencing kit (PE Applied Biosystems). Methylation was measured usingrestriction enzymes XbaI/SacII in CpG islands surrounding the TATA boxby Southern blot analysis.

5.2.1.10 Promoter Activity Assay

NOEY2 promoter fragment was amplified from genomic DNA by PCR™ andcloned into pGL2, a vector with luciferase reporter, its activity wastested by luciferase assay system (Promega, Madison, Wis.). A −750A to Gmutant construct was made by site directed mutagenesis (Stratagene).Mutation was confirmed by sequencing.

5.2.2 Discussion

The inventors expect that a loss of NOEY2 will be observed in asignificant fraction of advanced stage and high grade ovariancarcinomas. The inventors predict that NOEY2 may be lost in a smallerfraction of grade I and lower stage tumors. Moreover, the inventorspredict that loss of NOEY2 may be a rare event in borderline tumors, butthat this might correlate with high risk of recurrence. Given theeffects of NOEY2 on the growth of ovarian cancer cell lines, theinventors predict that the loss of NOEY2 may correlate with a poorprognosis in frankly invasive lesions. Although the inventors predictthat loss of NOEY2 may correlate with conventional risk factors, NOEY2may be of even greater interest if its loss correlates with shortenedsurvival but not with these conventional factors. Loss of NOEY2 may ormay not correlate with drug resistance. If this were the case for somedrugs and not for others, the marker might prove particularly valuablein choosing primary therapeutic regimens for individual patients. Lackof a correlation with drug resistance would suggest that loss of NOEY2was a marker for aggressive biological behavior. Interaction with otherbiological markers may be of particular interest. If NOEY2 inhibitssignaling through the Ras pathway the inventors would anticipateamplification of the poor prognosis associated with persistence of EGFR,overexpression of HER-2 and novel expression of fms. In contrast totumors at other sites, the inventors' studies with ovarian cancer havedemonstrated a high correlation between mutation and overexpression ofmutant p53 protein. Considered as a single marker, overexpression of p53has not correlated consistently with prognosis in ovarian cancer. IfNOEY2 plays a role in apoptosis, the mutation and consequentoverexpression of p53 might be a poor prognostic marker in that subsetof ovarian cancers that have lost NOEY2.

5.3 Example 3 Mechanism of Loss of NOEY2 Expression

NOEY2 has been mapped to 1p31. The inventors' data for 6 highlypolymorphic markers located at or near 1p31 indicates that LOH occurredin 5 of 12 informative tumors from matched pairs of ovarian cancer andperipheral blood samples. This rate of 42% loss is higher than thatexpected given the approximately 20% LOH rate throughout the genomeobserved in advanced ovarian cancer.

Using the 1p31 chromosomal localization and a series of 8 currentlyavailable highly polymorphic markers for 1p31, the inventors mayundertake LOH analysis of the region around NOEY2 in DNA from ovariancancers and normal peripheral blood leukocytes of the same patients. Inaddition to the inventors' own series of approximately 25 matched pairscurrently under analysis, the inventors may assess 100 matched pairs oftumor and normal peripheral blood leukocytes with histological andoutcome evaluation. The inventors may correlate LOH at 1p31 with outcomeas well as with stage, grade and histology.

Southern blotting analysis has failed to detect abnormalities in thestructure of the NOEY2 gene in 6 of 6 ovarian cancer cell linesassessed. The inventors may extend these studies to include the ninefreshly isolated ovarian cancer ascites samples from patients which theinventors have already demonstrated do not express detectable amounts ofNOEY2 RNA. If, as expected, the NOEY2 gene is intact in the freshlyisolated samples as it is in the already characterized tumor cell lines,it is most likely that the loss of NOEY2 expression is due to changes inmethylation status rather than due to deletion or to a transcriptionalmechanism.

As noted above, hypermethylation is a relatively frequent mechanism forloss of expression of tumor suppressor genes such as VHL, p16 and RB.The inventors may thus assess the effect of decreasing methylation with5-azacytidine or 5-aza-2-deoxycytidine on expression of NOEY2 in ovariancancer cell lines. The inventors may also assess whether the promoterfor NOEY2 contains CpG islands and is hypermethylated in ovarian cancersas compared to normal epithelium. Alternatively, mutations might befound in the promoter region which may be sequenced in those cell linesand tumors where downregulation of NOEY2 expression cannot be attributedto other mechanisms.

5.3.1 Methods

LOH analysis is performed with 8 commercially available highlypolymorphic markers for 1p31 using techniques published previously. Theanalysis of the 100 samples utilizes a semi-automated microtiter systemfor analysis providing rapid throughput for the 100 samples and 8 primerpairs.

Southern blotting is performed using standard techniques on DNA isolatedfrom highly purified (>95%) ovarian cancer cells isolated from theascitic fluid of ovarian cancer patients and tumor cells isolated fromsolid tumors (samples with >80% tumor cells).

Hypermethylation of promoter regions may be sought in ovarian cancersthat lack NOEY2 expression without LOH or an evident abnormality onSouthern blots. Ovarian cancer cell lines demonstrated not to expressNOEY2 and shown to have a relatively intact NOEY2 gene on Southernanalysis are cultured for 48 h to 2 wk in 5-azacytidine (5 M) or5-aza-2-deoxycytidine (0.75 M) to determine if this alters theexpression of NOEY2 as assessed by Northern blot analysis.

The inventors contemplate two BACs may be most appropriate for promoterisolation. Analysis of the NOEY2 promoter is conducted to determinewhether the promoter for NOEY2 contains CpG islands. The methylationstatus of the promoter for NOEY2 in ovarian cancer cell lines isassessed by restriction analysis with methylation-sensitive restrictionenzymes including HhaI, NotI and SacII. Alterations in enzymesensitivity may be due to changes in methylation, and this is analyzedby comparing the restriction patterns with those of normal ovarianepithelial cells which express NOEY2 and with ovarian cancer cell linesincubated with 5-azacytidine or 5-aza-2-deoxycytidine.

To determine whether any CG rich regions in the promoter of NOEY2 arehypermethylated in cells in vivo, ovarian cancer cells may be isolatedand purified from ovarian cancer patient's ascites. Ovarian cancer cellscan be purified to over 95% homogeneity (Hurteau et al., 1994) and mayroutinely survive in culture for at least one month. Such cells areassessed for the effect of 5-azacytidine or 5-aza-2-deoxycytidine onNOEY2 expression and restriction analysis with methylation sensitiverestriction enzymes including HhaI, NotI and SacII as described for celllines above.

If the methylation sensitive restriction enzyme approach described abovefails to detect methylation of the NOEY2 promoter in freshly isolatedcells, a method for methylation detection may be utilized which dependsupon the chemical modification of cytosine to uracil by bisulfitetreatment (Frommer et al., 1992). This technique is less sensitive tocontamination of tumor by normal cells and is more sensitive indetecting hypermethylation than is the restriction enzyme approachdescribed above. In this method, genomic DNA is bisulfite treated andfragments from within the promoter containing region cloned using PCR™and the TA vector and then subjected to sequence analysis. As bisulfitetreatment converts unmethylated cytosines to uracil, but leavesmethylated cytosines intact, sequence analysis may reveal the conversionof unmethylated cytosines to thymidines, while methylated cytosines mayappear as cytosines in the sequencing analysis. Sequencing of a numberof TA vector plasmids from tumors may demonstrate the frequency ofmethylation of the NOEY2 promoter.

Transcriptional regulation may be evaluated in those cell lines andtumor specimens where loss of NOEY2 expression cannot be attributed toother mechanisms. At least 500 bp of the NOEY2 promoter may be clonedfrom the BACs already identified that contain the NOEY2 coding sequence.The promoter may be ligated to luciferase as a reporter gene (Gum etal., 1996). Transient expression is sought after transfection of ovariancancer cell lines that lack NOEY2 expression and, as a positive control,normal ovarian surface epithelial cells that express NOEY2.

5.3.2 Discussion

The inventors' data suggest that more extensive LOH analysis of 1p31 maydemonstrate LOH for 1p31 in more than one third of ovarian cancers. Theinventors predict that the NOEY2 gene may be intact in ovarian cancersas suggested by Southern blot analysis and that the NOEY2 promoter maybe hypermethylated in a fraction of tumors when compared to normalovarian epithelium. If sought, mutations may well be found in a fractionof those promoters that are not hypermethylated. Transcriptionalregulation may be the least frequently occurring abnormality, however,cell lines that exhibit such an abnormality are valuable for elucidatingmechanisms of physiologic as well as pathologic regulation of NOEY2expression.

5.4 Example 4 Effects of NOEY2 Expression on Ovarian Cancers

NOEY2 expression is consistently lost from ovarian cancer cell lines andfrom freshly isolated cancer cells from patient's ascites. Further, dataindicate that expression of NOEY2 decreases clonogenic activity ofovarian and breast cancer cells in a transient transfection assay. Thissuggests that NOEY2 may alter characteristics of ovarian cancer cellswhich could lead to initiation or progression of ovarian cancer. Theinventors contemplate that upregulation of NOEY2 may inhibit growth,block invasion, reduce metastatic potential, decrease angiogenesis ortrigger apoptosis, whereas downregulation of NOEY2 may stimulate growth,increase invasive potential, augment metastatic potential, enhanceangiogenesis or inhibit apoptosis. Ras and Rap proteins requireappropriate localization and an intact effector domain to mediate theirfunctions. Mutagenesis of any of the amino acids in the CAAX box or theYDPTIEDSY domain impairs the ability of Ras to transform cells.

Conditional expression of normal NOEY2 is used to test whetherupregulation of NOEY2 inhibits growth, blocks invasion, reducesmetastatic potential, or decreases angiogenesis, and uses a dominantnegative NOEY2 (N17-NOEY2 modeled on other dominant negative small Gprotein constructs) to determine whether downregulation of NOEY2activity may stimulate growth, increase invasive potential, augmentmetastatic potential, or enhance angiogenesis. As transient expressionof NOEY2 inhibits colony forming activity, the inventors assess whetherexpression of NOEY2 may alter cell cycle progression or direct cells toapoptosis.

Growth inhibition by a number of mediators including heregulin canrequire an intact p53 pathway or an intact RB pathway. Approximately 50%of all ovarian cancers have mutations or deletions in p53. All of theovarian cancer cell lines thus far assessed for growth inhibition byNOEY2 contained normal p53. Although mutations in RB or p16 are rare infreshly isolated ovarian cancer cells, the inventors have identifiedovarian cancer cell lines with deletions or mutations in p53, deletionsin p16 and cell lines with deletions in RB.

The effector domain of Ras is required for the transforming activity ofthe protooncogene. The effector domain of Ras and Rap family members iscritical for Ras and Rap functions. The effector domain of NOEY2 differsfrom that of Ras and Rap family members. Site directed mutagenesis maybe utilized to convert the effector domain of NOEY2 to the identicalsequence to that found in Ras and Rap family members.

5.4.1 Transient NOEY2 Expression

Normal and epitope-tagged NOEY2 has been inserted into CMV and SV40promoter driven expression constructs. In order to distinguishtransfected NOEY2 from native NOEY2 and to demonstrate expression ofNOEY2, the NOEY2 gene has been epitope tagged with an hemagglutinin (HA)epitope using a triple HA repeat plasmid. The HA epitope was added tothe N terminus of NOEY2 so as to be distant from the potentialmyristylation site at the C terminus of NOEY2.

5.4.2 Conditional Expression

Ovarian cancer cell lines that lack NOEY2 expression may be transfectedto provide conditional expression of NOEY2. Studies demonstrated thatstable NOEY2 transfectants could not be developed likely becauseconstitutive NOEY2 expression suppresses cell growth and/or causes celldeath. The Tet-Off and Tet-On Gene Expression System provides regulated,high-level gene expression as initially described by Gossen and Bujard(1992). In the first transfection, the Tet-On regulatory protein isintroduced into ovarian cancer cell lines by transfection of a“regulator plasmid”-pTet-On (cells already isolated). NOEY2 and epitopetagged NOEY2 have already been inserted into the pTRE (plasmid created)which may be introduced into cells expressing the Tet On plasmid tocreate double-stable Tet-On cell lines which may express NOEY2 only inthe presence of tetracycline. NOEY2 gene expression should be negligiblein the absence of tetracycline and induced by the addition oftetracycline. The Tet-on system has the advantage over most induciblesystems in having very low levels of non-induced expression (leakiness)and in being compatible with induction both in vitro and in vivo.

5.4.3 Colony Formation

The ability of mutation of NOEY2 to alter its activity in colonyformation activity may be assessed as described. Mutant or normal NOEY2may be transfected into ovarian cancer cells along with a hygromycinresistance plasmid. Cells may be incubated with hygromycin for 2–3 wkand colonies assessed by staining with 1% Coomassie blue in 30% methanoland 10% acetic acid.

5.4.4 Proliferation, Invasiveness and Apoptosis

Proliferative capacity may be judged by ³H-thymidine incorporation,growth by MTT assay and clonogenicity by growth in soft agar in thepresence and absence of tetracycline. Invasion of matrigel membranes maybe studied using techniques already established in the inventors'laboratory (Berchuck et al., 1992). Apoptosis may be evaluated bymorphology, free DNA ends (Apoptag kit), DNA ladders and loss ofmembrane asymmetry (Annexin V staining).

5.4.5 In Vivo Tumorigenicity

The methods for murine tumorigenicity assays have been described.Briefly, the parental ovarian cancer cell lines-SKOv3 and Hey aretumorigenic in nude mice (HEY forms subcutaneous tumors whereas theHEY-A8 cell line forms intraperitoneally tumors and metastasizes). Fiveto 10×10⁶ stably transfected tumor cells from each cell line may beinjected subcutaneously and intraperitoneally into athymic nude mice.All recipients should develop tumors within 2 wk. If the behavior oftransfected cells reflects the parental phenotype, tumor cells may formnodules, ascites, and metastasize to the lungs where nodules can bedetected grossly and by histologic examination. However, whendoxycycline is given to the mice at a dosage sufficient to establish anappropriate doxycycline concentration, NOEY2 gene expression in thetumor cells should be upregulated and growth inhibited. Doxycycline canbe delivered either orally as a glucose suspension or optimally as along release pellet (Innovative Research). Subcutaneous tumors may bemeasured twice a wk and animals examined every other day for ascites.

5.4.10 Discussion

NOEY2 inhibits colony forming activity of breast and ovarian cancer celllines. The effects of NOEY2 may be expressed at many levels byinhibiting proliferation, colony formation, anchorage independentgrowth, invasiveness and ability to form tumors in nude mice. The globalinhibitory activity of NOEY2 may extend to an ability to blockproduction of growth factors and angiogenic factors such as VEGF. NOEY2may thus decrease the ability of tumors to grow in the peritonealcavity, to metastasize and to enlarge due to lack of neovascularization.Data indicate that even 48 h following transfection of NOEY2, there aredecreased levels of reporter constructs and markedly decreased numbersof transfected cells as indicated by contransfection of the GFP plasmid.This suggests that NOEY2 expression is likely to induce apoptosis.

5.5 Example 5 Mechanism of NOEY2 Interference with Signal Transduction

Data indicate that NOEY2 appears to function as a tumor suppressor inovarian cancer. NOEY2 could function as a tumor suppressor byinterfering with signal transduction from surface receptors, byinterfering with signaling cascades, or by altering the function orexpression of proteins required for cell cycle progression.Alternatively, NOEY2 may target cells for apoptosis. This specific aimmay attempt to identify the site at which NOEY2 mediates it tumorsuppressor activity.

By analogy with members of the Rap family with which NOEY2 sharessimilarity in the effector domain, the product of the NOEY2 gene may actas an antagonist of Ras p21 protein signaling. Ras provides aconvergence point for signal transduction induced by tyrosine kinaselinked and G protein linked receptors. A major pathway activated byp21-Ras is the Ras-Raf-MEK-ERK (MAPK) module which plays an essentialrole in cell growth and differentiation. Due to its structuralsimilarities to p21-Ras, NOEY2 protein may compete for the upstream ordownstream regulators and effectors of Ras. This is a suggestedmechanism of action for the ability of Rap family members to decreaseRas-mediated signaling.

By analogy to members of the Rac family with which NOEY2 has homology inthe effector domain, the product of the NOEY2 gene may act as an agonistof the JNK pathway. Similar to the p21-Ras MAPK cascade, the JNK pathwayincludes a similar G protein-cascade module,RAC-MEKK1-SEK/MKK4-JNK/SAPK. In contrast to MAP kinases, JNK has beenlinked to stress response, growth inhibition and programmed cell death.Since the JNK pathway is a negative regulator of cell proliferation, anagonistic activity of NOEY2 in this pathway could explain the tumorsuppressor-like activity of NOEY2. Thus it is possible that NOEY2protein inhibits cell growth through activation of the JNK pathway.

Since NOEY2 decreases colony forming activity, it is likely that itinhibits cell cycle progression. The inventors may assess whether intactp53, p16 or RB are required for NOEY2 to inhibit colony forming cellactivity. If the p53 or p16/RB pathways are obligatory for NOEY2inhibition of colony forming cell activity, the inventors may assess theeffect of NOEY2 on activation of these pathways.

As an alternative to inhibition of cell cycle progression, NOEY2 maydecrease colony forming cell activity through induction of apoptosis. IfNOEY2 increases the rate of apoptosis, the inventors may assess theability of NOEY2 to alter expression of members of the Bcl-2 family ofproteins which play a major role in the regulation of programmed celldeath in ovarian cancer cells according to the inventors' data.

Small G proteins are active in the GTP bound state and inactive in theGDP bound state. Several of the amino acids present in NOEY2, if presentin Ras would result in activating mutations. One may assess whether theGTP/GDP ratio of NOEY2 is similar to that of normal Ras in restingfibroblasts or whether it is similar to that of activated Ras. Thelocalization of Ras and other small G proteins through myristylation iscritical for their function. One may determine the localization of NOEY2in ovarian epithelium. Because NOEY2 may function at a number of levelsin suppressing tumorigenesis, it may be interesting to determine whetherNOEY2 inhibits the transforming activity of growth factor receptors(EGFR, HER-2), intracellular tyrosine kinases (src), activated Ras anddownstream mediators of Ras (RAF).

It has been well established that stimulation of tyrosine kinasereceptors (with EGF) or G-protein coupled receptors (withlysophosphatidic acid) activates MAPK through the Ras-Raf-MEK-ERKpathway. If NOEY2 expression indeed impairs EGF- or LPA-induced MAPKactivation, further studies using activated Ras, Raf or MEK may beconducted to determine where NOEY2 protein inhibits MAPK activation,e.g. upstream or downstream of Ras. Expression of activated forms of Ras(V12 Ras), Raf (v-raf) or MEK causes constitutive activation of MAPK.Expression plasmids carrying V12 Ras, v-raf or MEK may be cotransfectedwith NOEY2 or an empty vector. MAPK activity in transfected cells maythen be determined using an epitope-tagged cotransfected MAPK. Acomplete or partial inhibition of Ras-, Raf- or MEK-stimulated MAPKactivation by NOEY2 would be expected if NOEY2 inhibits the Rassignaling cascade.

The RAC and CDC42 small G proteins activate the JNK signaling pathwaywhich negatively regulates cell proliferation targeting cells toprogrammed cell death. As the effector domain of NOEY2 has a sequencewhich is similar to that of RAC and CDC42, the inventors may assesswhether NOEY2 activates the JNK kinases. The inventors may also assesswhether NOEY2 is required for activation of the JNK kinases by TNF, IL1,FAS, lysophospholipids, and protein synthesis inhibitors (anisomycin)all of which the inventors have shown activate JNK kinases in ovariancancer cells or activate ovarian cancer cells and have been demonstratedto activate JNK kinases.

The inventors may assess whether intact p16 or intact RB is required forNOEY2 to mediate inhibition of colony formation. If p16 or intact RB isrequired for the inhibition of cell proliferation, the inventors mayassess the effect of NOEY2 on p 16 and cyclin D1 levels, on CDK4 kinaseactivity and on RB phosphorylation status. The inventors may also assesswhether normal p53 is required for NOEY2 to mediate inhibition of colonyformation. If p53 is required for NOEY2 to mediate inhibition of colonyformation, the inventors may assess whether NOEY2 alters expression orstability of p3, expression of cyclin D1, CDK2 kinase activity andexpression of the P21/WAF1/CIP mediator of p3 action.

5.5.1 Methods

The inventors have developed polyclonal (9613, 9617) and eightmonoclonal (17G6, 15E11, 12C7, 6B4, 6D2, 12A8, 3H2 and 16C6) antibodiesto NOEY2 with techniques that the inventors have successfully utilizedin the past. The inventors have tagged NOEY2 with HA-epitope allowingthe inventors to distinguish transfected NOEY2 from endogenous NOEY2.Until polyclonal or monoclonal antibodies specific to NOEY2 areavailable, the inventors may utilize transfection of epitope-taggedNOEY2 to demonstrate function. The HA antibody (for which the inventorshave access to the hybridoma) immunoprecipitates tagged proteins andworks well in Western blotting.

GTP/GDP ratios NOEY2 GTP/GDP ratios are assessed using ³²P0₄-loadedcells as reported previously for determining Ras GTP/GDP ratios (Kruk etal., 1990). NOEY2 may be immunoprecipitated and washed. GTP and GDP maybe separated by thin layer chromatography and ³²P-containing GTP and GDPidentified by autoradiography and comparison with ninhydrin stainedstandards.

5.5.2 NOEY2 Localization

Normal ovarian epithelial cells which contain more NOEY2 protein thanovarian cancer cell lines may be used. Cells may be labeled with³⁵S-methionine for 3 h and nuclear, cytoplasmic and membrane fractionsprepared by differential centrifugation. Equivalent amounts of eachsample may be immunoprecipitated and analyzed by SDS-PAGE. To complement³⁵S-methionine labeling, NOEY2 may be western blotted to determinelocalization. If insufficient NOEY2 is present to allow direct westernblotting, NOEY2 may be immunoprecipitated from each cellular fractionand localization determined by western blotting of each fraction. NOEY2may be identified by immunoprecipitation of transfected HA epitopetagged NOEY2 until such time as NOEY2 antibodies are available. Normalovarian epithelial cells can be effectively transfected.

To complement subcellular fractionation, HA may be immunolocalized usingconfocal microscopy and immunoelectron microscopy. The HA antibody andepitope tagged NOEY2 cannot be utilized for this purpose due tocross-reactivity of the HA antibody with cellular proteins. Thesestudies may be performed when appropriate affinity purified polyclonalor monoclonal antibodies are available.

5.5.3 Target of Signaling Inhibition

NOEY2, antisense NOEY2 and dominant negative NOEY2 may be cotransfectedat a 5:1 ratio with activated erbB, HER-2, src, Ras and RAF into NIH 3T3cells to ensure that NOEY2 is present in all transfected cells. Theeffect of NOEY2 on focus forming activity induced by each oncogene maythen be assessed. The inventors have NIH3T3 cells in the laboratory witha low baseline focus forming rate which are appropriate for this assay.NIH 3T3 cells constitutively overexpressing myc are available, which maybe used to determine whether NOEY2 may inhibit the focus formingactivity of these oncogenes in the presence of high levels of myc.

5.5.4 Signal Transduction

The ability of NOEY2 to alter activation of Ras, MAPK, or JNK may beassessed by cotransfection of NOEY2, antisense NOEY2, and dominantnegative NOEY2 (not epitope tagged) and an epitope tagged target.Alternatively if conditional tetracycline-induced NOEY2 containing celllines are developed, the inventors may study endogenous targets with andwithout Tet induction of NOEY2 expression. The inventors may also assesswhether NOEY2 affects the ability of activation of tyrosine kinaselinked receptors (EGF and heregulin), activation of protein kinase C(phorbol esters) and G protein linked receptors (lysophosphatidic acid)to stimulate the activity of these signaling molecules. Although Ras andMAPK activity are elevated in several of the cell lines under study, Rascan be incrementally activated by each of these agents in ovarian cancercell lines (Patton et al., 1994).

GTP/GDP ratios on Ras may be determined as described above with ³²Plabeling and immunoprecipitation of epitope-tagged Ras with the anti-HAantibody or in conditional NOEY2 expressing cells with the Y13-259anti-Ras antibody (hybridoma in house).

5.5.5 MAPK Assay

The effect of NOEY2 on MAPK activation may be assessed by fourassays: 1) gel mobility shift. 2) Western blotting with an activationspecific epitope antibody (UBI) 3) in gel kinase assay (the epitopetagged MAPK runs at a different size from the native MAPK) and 4) invitro kinase using MBP as a substrate (Xu et al., 1994).

5.5.6 JNK Assay

JNK activity may be determined by in vitro kinase assay using aGST-N-terminal portion fusion protein of Jun as substrate. The inventorsmay also assess whether expression of NOEY2 or of dominant negative (N17NOEY2) may alter anisomycin, TNF, IL1 or FAS induced activation of JNK.

5.5.7 CYCLIN D1 Expression

If conditional tetracycline-induced NOEY2 containing cell lines aredeveloped, the inventors may study endogenous cyclin D1 levels with andwithout Tet induction of NOEY2 expression. Alternatively, if conditionalNOEY2 containing cell lines are not available, the inventors may utilizecotransfection of NOEY2 and a cyclin D1 luciferase expression constructas a reporter.

CDK4 and CDK2 kinase activity may be assessed followingimmunoprecipitation using recombinant RB (UBI) as a substrate.p21/WAF1/CIP1 and p16 levels may be assessed using western blotting withUBI antibodies. RB phosphorylation may be measured by gel shift assayusing RB specific antibodies (UBI). p53 levels may be assessed byimmunohistochemistry and western blotting. Although p53 levels arenormally low, they can be increased by transcriptional or posttranscriptional mechanisms which can be detected by an increase inlevels as detected by these methods. The inventors may assess the effectof NOEY2 on expression of these proteins by western blotting or byimmunoprecipitation followed by western blotting where protein levelsare low.

Sequence analysis of NOEY2 predicts that NOEY2 may have constitutivelyhigh GTP/GDP ratios. As NOEY2 contains a CAAX Box, the inventors predictthat NOEY2 may be primarily a membrane associated protein. The inventorshave demonstrated that NOEY2 inhibits the growth of ovarian cancer cellswhich contain activated or mutant Ras. The inventors predict that NOEY2may function similar to Rap family members and may inhibit activation ofthe Ras by tyrosine kinase, and G protein linked receptors. Theinventors predict that NOEY2 may inhibit MAPK activation induced bytyrosine kinase and G protein linked receptors and by activated Ras. Byanalogy, the inventors predict that NOEY2 may not inhibit activation ofthe Ras pathway by activated RAF or MEK. The inventors predict thatNOEY2 may function at the G1 phase of the cell cycle. The inventorsexpect that NOEY2 may block cell cycle progression in G1 by inhibitingthe pathway leading to RB phosphorylation. Without further data, it isimpossible to predict at which level in the CDI/CDK/Cyclin RB cascadethat be regulated by NOEY2.

5.6 Example 6 Genomic Sequence of NOEY2 (SEQ ID NO:5)

FIG. 7 shows the genomic structure of the NOEY2 locus. The genomicsequence of this region appears below:

NAGAANAGGGTCCAAGGNGTGGGAGAATAGNTTGTGTANACATTGNAGGAAGACNGAAGATACAGGCAGAACATCNGTCAATAGAGGGNAGGGANAACATGGGTTTGACCNGGAAAGCCGGTACATTTNGGAGGAGGAGGGGTNTGGCCAGTGGTGGCAGGAGGGAGGTTGTGGAAGCCAGGGTTCTTGTTACATATGNGAAGCCTGTAATATGCTTCAGAAAGAATAGANGGCATATGTACCTCAAAAGGTAAATGACCTTAAACGGTGTCAGACTNTNAGTTAAATCTCTCCCGGATCAGAGAAAGACCTGGAAAGGGAAGGAGATTGTCCACAGAACACAAATTTCCCTCGCAAAAGATAGCATTGCACAGGACCATTCCAAAATATGTCAGAAATATATTNTGGGGTAAAATACTTTGATCGCCCTTAGGGCTGNTACCTGTCATGTGATGCTATACCAGAATCAGGTTGGAATTTGTTTTGAGACAGGTTCTCACTTTGTTGCTCAGATTGGAGTACAGTGGCAGTGATCACGGCTCACTGCAGCCTCGATCTCCTTGATGTGGGAGGCTCAAGTGATCNTCCCACATCAGCCTCCCAAATAGCTGGGACTACAGGCGGCCACCACCACACTGGGCGATATTNTTTAAAAGTAGAGACAAGTTCTCCCCATGTTGCCCAGGCTGGTNTNTAACTCNTGGCCTCAACCTCCTTATTTTNTAGGATTACAGGCGCCAGGNTAGACCTCACAGGTCTTTAGACTTTTACGCACCAGGTACCTGGTAGGGGGAGGGATTATAGTGGCAGAAGAGCAGTACCAGTGGCCCACACCACACACCCTGGCNTCAGCTGGCTGGGGCACACAAAACCAGGTGCTACCGTCAACGACTAGGCCCATAGGGTACTGCTGTCAAAACCTGCTGCCAACAANTTCCACACACTCCCCAAAACGCTCGGTAGGCGGTGGTGCGCAGCTTTCAATGCATCCGCCGCCAGGCGCTCACAGGCAAGGGAGAAAGAAGCCAGACGGAGCTCGGAGATGTGGAGGGCAGACGCAGGCGCATTTGGAAAAGGGACTGGCGGTGGGAGGCGCAGAGGGAAAAAGGAACGACACAATCGGGCTTCNTAGCCGCTGGCGGACCCGATGGGGCGTCCTGCGAGGGTTCGGCGAGGGTTCTGCCAGGATAGTAGCATTGCGCCCAAGGAGTGAGAGGCACCCGGGGNTACTGGAGCCAGACCCTCAACCCGCCCTAGTGGGAGGCGAAGAACAACGAAAGCCTCGTATTCCCATTTCTNTAATGGCTAATGACATTAAAGGTTTTCATATGGTTATTTGCCATCTGCATATCTTCAGTGAAATGTCTGTTTATGTCTTCTGACTATTCTCTAATTGGATTTTTAAAAATAATTGGTTTTTGAGTTATTTACATATTCTAGATACTGGTTGTATGCCAGATATATGGCTTGTAAATATTTTCTCCTAGGTAAACCTTTTCAGTATCGTTACAGGGTCTTTCACAAAGCAAAAGTTTTAAATTTATGGAGTCTAATTCATCAACATTTCTTTTTACCGGTCTTGCCACTAATGTCAATTTAAGAACCTTTTGCCTAGCCTTAGACAATAGTTTGTTGTTTTTTAAAACCGTTTTGTAATTTTACTCGTCACAGTTATATCTAGCCATTAATTTTTATGTAAGGGTTATTTTTGGCGGGGGGGAATNTATAGATGTCCTGTCTTTTCAGTATCATTTGTGGAAAAGATTATNTGTCCTGCATTAAANTGCTTTTGGACTTTNGTCTAAAATCAGTTGGACCGGTTTTTGTTGGCAAAGTTTTGCCTGAAGCTTATTCCAACAGGTGAGAAAAAGTCCACAGTTTAACAGTTCNTCCCCAACCTGTAACCCCGCCTTGAACTTTTGGAATAGCCCCTCGATTGTTGTAGATGCCAAGCGGACCTCGCGCCGCTNTGCGTTGGGCCAGCCCCTCACAGCTGGTTTNTTACCANGTATTGCGCAAGCGGAATTTATGCNTGTTACCCACACTCCNTGCGCCCCCGCACCCCGNTCCTGTGCGCAAGTCGGAATATAAAACCGCGGAGGAGTGAGCTCTTGGGGTGTCCAGTTGGTTGCCGCGGCAGTCTCTCCGAGCAGCGCATTTGTCTTCTAGGCTGCTTGGTTCGTGCCTCCGAGAAAGGTAAGTCTTTCTTTCGCTTTTTTAGGGGTACTTGAAAACAACAAGTGTCAGACAAAGCAGCAGATGCTGTTGCGCAGTANAAGTTTATGGGCGAGTTGTCCCTGAAACTGGAACCAGGTCTTTCTTGGCGCGATTACGCAAGAACCACCCGCAGCCCTGCGGGCTCCTGGCAGGTCCTGCAACTGCACTTTGGATAGTCCCGTTGGGAAGCTAGCACTTTTTAATATAAAAGAACGAGGTTTGATAAGTGTGCGAGCTTAAAGGTTGACACAGTGTCCACTATTACAGCTGCGTANGTAGCTAGTGTTCAGGAAGTAATAGTGGAGTCATGTAGTGTGAAAGTAAGATTGAAATGGGCGAGGAGGGTAGCAGCCGCCACAGCCACCAGAGAGAAACCTGACCTTGCAGGTGCGTGGTGATGTCCATGAGCCAGGCTGGTGCCGCAACAGCAGCGGCGGGACCTTGAGCTCCGCACGGCCGCTGGGTTTGGACGCCCTCTGGTTCCTGGAAACTTTCACCTCCCCCTCAGCCTGAGGCCAGGTGGCCTGGGAAGGTGGAACGAGTGTGGAGGGGAGTGGGGGGGGGGGTCCACTGCCTGANAATGANAATTCTCTTCACATCTGGAAATTCAGTTATCACGTGTGTCCTTTACCAATTTTTTTTCTTTTATTTTCTTTTTGATAGAGACGGCGGTCTCCCTATGTTACCCAGGCTAGTCTTGAACTCCTGTGCTTAAGCGATCCTCCCACCTTGACTTCCCAAAGTGCTGGAATTACAGGCATGAGCCAATGCGCCCGGCTGCTTTACCAATTTTCTATGAATGAATTTGTACATACATCCCCTAGANCAGGAAGTNATGTANAACAGAATAATTAGTAATGCACATTTCCTAATGTGGGATGTTGGTGGCCNACAGATATTTGGTCTTTACTGGAACTCTTGATACTAACATGGNAGTTTATAATAGTTGTGGAGAGTGCAGACAAGGCTAGGATTTCTGTGACTAGAGAACTCTTAGTGCGTGAAGACCTAAGGAAAGCTGGATTGTTGATTTTTGTTAATAAATAAGATGTGAAAGATTGCATCACTGTAGCAGAAATCTCCTAGTTTTTTAAGCTAAATTCTATTAAAGGTCATCATTGCTAAAGGAATTGTGCCCAGGATTTGGATAGCTGATGTCATTACTTAATATTAGATGATATCAACTAACCACATCTCATAGACTGGAATAAAGTGCTAGATTTTACCTGAAAGCTGCAAAAATGAATGGTTTAGATATATGTATGTATTTATTTTATATCAATTTCAAATATTTACTGTATTAACCTCCCTGGCCCCCTTTAATCAAGAATATAAAATCATCTACTTAAATTTTGCCACTTAAGTTTAGAACACTCTTAGAATCACACTATCTTAAAGAAGCCAGACTAGAATTAGAAGCAANTTAANTCTGAAGATATAACAACCAGCAACAACATTTTTTTTTTTCAAATGAAAACTCTAATATGGGGTGGGTATGTTGTGTCACACCTGTAATCCCAACACTTTGGGAGGCTGATGCAGGAGGATCACTTGAACACAGGAGTTCAAGACCANCCTGGACAACATAGCAAAACCCTGTCCCTACAAAAAATAANAAAATTAGCTGGGCATGGTGTCACATGCCTGTAGTCCCAGCTACTCGGGAGGCTGAGGTGANAGGATTGATTGATCCCAGGAGGTTGAGGCTGCAGTGAGTCATGATCGCATGACTGCACTCCAACCTGAGGGACANAGCAAGACCCTGACTCAAAAAAAAAAAACAAAAAAAAAAAAACCACCACCAAAACTCTAATATGGACATATTACTCTCTCATGGGACTTGCACATTCTAAAAAGGGTCCTTTTCCCCAGTACTGGGANANTATNTGTTCAACTACGCAGCCAGCAANACAGGCTATTTTATATAGGGAGTGTGCTATTCACAAAAAGCCTCTCTTCTCTTTCTGGTATTGTACATGACACAATCATAGCTGTACCTGAAAAAAANTGCATTTTAAGGACCATCATCACCTAAAAACATGTNTAAATTTCTATACCTAGTGCCACAGGAATNACATTGCCTTGTACTATTCCTACCTCTGTCCAAAGGCCAGCTATGTGGTCTGTCTGCATGGTGCCTAAAACTTTTTCCATCTGACCTAGGATGCTTCTGAAGCAGTCCCCCTGGGCAGCTGTCTGGTATTTAGGATATACCTGTGAGAAAANTTNCTTACAACCCTAATCTACTATGTTTATTCCTGAACTCAAAAANTTCATTTGACTGTTCAATTCCTGAAATTTNCTCTATTTCCANAAGGCTGAATTAAAATTACTTTGTTAAAGGTANTAGCCATGGCAAAAAAAAAACCACTGTTCTGTAAAAAAACTCATTCAATATTTACAATCTTTTCTAATCAAAAATTANATCCTGAAAAGAAAGGTTCATATATATATATATATATATATATATATATATATATATATATATATATATATATCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTACTCCACTGTCATTGTGACTAAGGATTCATGAACTAAGACCCCTCCCTCAGCTTTTGGTGGCACATGGTGACAGCATGCTCAGAGCAAAGGTGCTCCCCATGCCTNTTCTGGGGNTGCACTGACTGCAGGTACCTCCCCTTTTTACATCCCACACCAGTGAATCCAAAAACCCCCTCCTTTCTCCTGTANTGATGACTCTGTAGCTTTAACCAGGGNGACGGTGTCACTNTAAATGTCACCTTGGCATTCAGCCCCATAGAGTGGGGAAAATTCCCTCACCTGTTTCTCTTTGACTGTTCAGTCCACTTCAATTAAAATCTTAATTTTACAAGCGAGGAAATGAGAGTGTTTCTTGTAGGGGTGTAGTGAGAATTTAATAAAACAGTTTAAGGAAAGAAAACAAAAGGTAGTATTGCTGCACTTTNTAGATGGTAAAAAGCAAACCACCATGTCTGTTTAATATATATCACCTGCTGGTCCNTCGGTCTAGCAGGCTGAACTGTGTGCCTGGGAATTTTTTTCTCGCTGTGTGCACCCCTTTACGTCACAGGGTGGATCTCTTCAGAGTCCATGNGGAGCAGCTGGCCAGGCTGACATGATCTGACAAGATTGTAGGTTACCANTACCATCTCTCACCGTCTCACTTTCTTCCTAGGGGTCTCCTGCTGCCAGCTAAGTGTGGGAGAACTTGTGCACGTATCTCCCCTCCGAATCCCAACGATGGGTAACGCCAGCTTTGGCTCCAAGGAACAGAAGCTGCTGAAGCGGTTGCGGCTTCTGCCCGCCCTGCTTATCCTCCGCGCCTTCAAGCCCCACAGGAAGATCAGAGATTACCGCGTCGTGGTAGTCGGCACCGCTGGTGTGGGGAAAAGTACGCTGCTGCACAAGTGGGCGAGCGGCAACTTCCGTCATGAGTACCTGCCGACCATTGAAAATACCTACTGCCAGTTGCTGGGCTGCAGCCACGGTGTGCTTTCCCTGCACATCACCGACAGCAAGAGTGGCGACGGCAACCGCGCTCTGCAGCGCCACGTTATAGCCCGGGGCCACGCCTTCGTCCTGGTCTACTCAGTCACCAAGAAGGAAACCCTGGAAGAGCTGAAGGCCTTCTATGAGCTGATCTGCAAGATCAAAGGTAACAACCTGCATAAGTTCCCCATCGTGCTGGTGGGCAATAAAAGTGATGACACCCACCGGGAGGTGGCCCTGAATGATGGTGCCACCTGTGCGATGGAGTGGAATTGCGCCTTCATGGAGATTTCAGCCAAGACCGATGTGAATGTGCAGGAGCTGTTCCACATGCTGCTGAATTACAAGAAAAAGCCCACCACCGGCCTCCAGGAGCCCGAGAAGAAATCCCAGATGCCCAACACCACTGAGAAGCTGCTTGACAAGTGCATAATCATGTGAGCCCTGGGCCTTAAGAGCCAGCTCTTCCTATCCTGTAGCGTGTAGAAAACGTGGACTCATTTCACTATGTTACATGTACATGGTTGATTTTGTGCTGTTGTTTGGACTGTAACATCCATGTTGTCAATACGTATACCTTGTAAGTGGATAACTTTTCTTTTTCCCAGGCCAGAGAATTCAAATTGTTAAAACATTGGCATTTGAAGAGGAGAACAAAATGTAGCATGATGTATTTAAAGTAAGGCCTTTAGTAATGAATGTAATGAGAGAAAATGTTTTGAAAAGAACAAAACATCAAAATGAATAGAAAGAAAAATTGGAAGGCGTCCTTTTGGTAACCCGATTATTGTGTATTACCTTTAAATATTTCACATCCTGTAAGTGCTTAATCATATCTTTTAATTGTGTATTTAAGAAAAGTGTTTTCACAACAAAAGCTTTTGATAAATTGCTGCGTGACATATACTAAATAAAAAAATGAATATGTTGATCATTAGGGGTGTGGGAGCAGAGAAAATTGTGAAAGTGACTCTCACTAAAGATGTTAGTAGTTTCTCATGTCATTTAAAAATGTTTGAGTATTCTGCATAGCAGTTTGTAAAAGTGTAACAGCTTATTGACTTAATAAAGCTTTTCCTGCATGCAATCAGCTGTAANAATTTGTCTCACCANAAAACAAAACATTGCCCATTGTATTAAAATTTAAACCATATCTGTTAAAAGTTTCCAATAAGAACTTCACACATGGATGTCCTTGCCATGTTGAAATTATCCAATATGGGAGGGGGGTGTTTTAGGGAGGTCTCTGCAATACANAGCTGTTTTGTGTCTTTCCTGAACTGACATCCCGAAAAACTCCAGGCATCTTTGAGGAAAATGGTCACAGTGTTGCTGTCTCANAGGAAGCGGGTGAAAAGCAAGCCTCTGCCTTCTGCCTCTTCCTATATTCTGAAATACTGGATATAGGCAATAGGGAGCAGAATGAAAGACAAGGGGAGGAATGATATTTGAGANACTCCCCCATAAGGGAGTTTTTAAAGAGATTATATTTGAACATAATTTTTTGAGCGAGGGAATAAAGTATACATATCCTTGCTTTTGANAGTTTTTTTTTTTTTTTTAAATTGGGAAANGTTCAGGGGAGGCNCTNAATCTANTGATTTTTTTCCCCCCNAAATNTTATTGAACNAATATCTATTGAACAATNTTNTNTNTTTCTACACAAAAANCACATTGTTCC

6.0 REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

-   U.S. Pat. No. 4,196,265, issued Apr. 1, 1980.-   U.S. Pat. No. 4,237,224, issued Dec. 2, 1980.-   U.S. Pat. No. 4,329,332, issued May 11, 1982.-   U.S. Pat. No. 4,489,055, issued Dec. 18, 1984.-   U.S. Pat. No. 4,554,101, issued Nov. 19, 1985.-   U.S. Pat. No. 4,683,195, issued Jul. 28, 1987.-   U.S. Pat. No. 4,683,202, issued Jul. 28, 1987.-   U.S. Pat. No. 4,800,159, issued Jan. 24, 1989.-   U.S. Pat. No. 4,800,159, issued Jan. 24, 1989.-   U.S. Pat. No. 4,873,191, issued Oct. 10, 1989.-   U.S. Pat. No. 4,883,750, issued Nov. 28, 1989.-   U.S. Pat. No. 4,913,908, issued Apr. 3, 1990.-   U.S. Pat. No. 4,965,188, issued Oct. 23, 1990.-   U.S. Pat. No. 4,987,071, issued Jan. 22, 1991.-   U.S. Pat. No. 5,176,995, issued Jan. 5, 1993.-   U.S. Pat. No. 5,225,341, issued Jul. 6, 1993.-   U.S. Pat. No. 5,276,269, issued Jan. 4, 1994.-   U.S. Pat. No. 5,279,721, issued Jan. 18, 1994.-   U.S. Pat. No. 5,334,711, issued Aug. 2, 1994.-   U.S. Pat. No. 5,354,855, issued Oct. 11, 1994.-   U.S. Pat. No. 5,384,253, issued Jan. 24, 1995.-   U.S. Pat. No. 5,451,410, issued Sep. 19, 1995.-   U.S. Pat. No. 5,482,852, issued Jan. 9, 1996.-   U.S. Pat. No. 5,500,224, issued Mar. 19, 1996.-   U.S. Pat. No. 5,508,468, issued Apr. 16, 1996.-   U.S. Pat. No. 5,550,318, issued Aug. 27, 1996.-   U.S. Pat. No. 5,556,617, issued Sep. 17, 1996.-   U.S. Pat. No. 5,620,708, issued Apr. 15, 1997.-   U.S. Pat. No. 5,631,359, issued May 20, 1997.-   U.S. Pat. No. 5,641,515, issued Jun. 24, 1997.-   U.S. Pat. No. 5,698,515, issued Dec. 16, 1997.-   Int. Pat. Appl. Publ. No. PCT/US87/00880.-   Int. Pat. Appl. Publ. No. PCT/US89/01025.-   Int. Pat. Appl. Publ. No. WO 84/03564.-   Int. Pat. Appl. Publ. No. WO 88/10315.-   Int. Pat. Appl. Publ. No. WO 88/10315.-   Int. Pat. Appl. Publ. No. WO 89/06700.-   Int. Pat. Appl. Publ. No. WO 89/06700.-   Int. Pat. Appl. Publ. No. WO 91/03162.-   Int. Pat. Appl. Publ. No. WO 92/07065.-   Int. Pat. Appl. Publ. No. WO 93/15187.-   Int. Pat. Appl. Publ. No. WO 93/23569.-   Int. Pat. Appl. Publ. No. WO 94/02595.-   Int. Pat. Appl. Publ. No. WO 94/13688.-   Eur. Pat. Appl. Publ. No. EP 0273085.-   Eur. Pat. Appl. Publ. No. EP 0360257.-   Eur. Pat. Appl. Publ. No. EP 92110298.4.-   Eur. Pat. Appl. Publ. No. EP 320,308.-   Eur. Pat. Appl. Publ. No. EP 329,822.-   Great Britain Pat. Appl. Publ. No. GB 2,202,328.-   “Manipulating the Mouse Embryo: A Laboratory Manual,” 2nd edition    (eds., Hogan, Beddington, Costantimi and Long, Cold Spring Harbor    Laboratory Press, 1994.-   “Modern Applied Statistics With S-Plus,” Venables W N, Ripley, B D    Springer-Verlag. New York, 1994.-   “Remington's Pharmaceutical Sciences,” 15th ed., pp. 1035–1038 and    1570–1580.-   Albanese et al., “Transforming p21^(ras) mutants and c-Ets-2    activate the cyclin D1 promoter through distinguishable regions,” J.    Biol. Chem., 270:23589–23597, 1995.-   Arshady, “In vivo targeting of colloidal carriers by novel graft    copolymers,” J. Mol. Recognit., 9(5–6):536–542, 1996.-   Baichwal and Sugden, “Vectors for gene transfer derived from animal    DNA viruses: Transient and stable expression of transferred genes,”    In: Gene transfer, Kucherlapati R, ed., New York: Plenum Press, pp.    117–148, 1986.-   Barany and Merrifield, “A chromatographic method for the    quantitative analysis of the deprotection of dithiasuccinoyl (Dts)    amino acids,” Ana. Biochem., 95(1):160–170, 1979.-   Baselga and Mendelsohn, “Receptor blockade with monoclonal    antibodies as anti-cancer therapy,” Pharmacology & Therapeutics,    64(1):127–54, 1994.-   Baselga, Norton, Masui, Pandiella, Coplan, Miller, Mendelsohn,    “Antitumor effects of doxorubicin in combination with anti-epidermal    growth factor receptor monoclonal antibodies,” J. Nat. Cancer Inst.,    85(16):1327–33, 1993.-   Bast, Feeney, Lazarus, Nadler, Colvin, Knapp, “Reactivity of a    monoclonal antibody with human ovarian carcinoma,” J. Clin. Invest.,    68:1331–1337, 1981.-   Bast, Jacobs, Berchuck, “Editorial: Malignant transformation of    ovarian epithelium,” J. Natl. Cancer Inst., 84:556–558, 1992.-   Bast, Klug, St. John, Jenison, Niloff, Lazarus, Berkowitz, Leavitt,    Griffiths, Parker, Zurawski, Knapp, “A radioimmunoassay using a    monoclonal antibody to monitor the course of epithelial ovarian    cancer,” New Engl. J. Med., 309:883–887, 1983.-   Benvenisty and Neshif, “Direction introduction of genes into rats    and expression of the genes,” Proc. Nat. Acad. Sci. USA,    83:9551–9555, 1986.-   Berchuck, Kamel, Whitaker, Kems, Olt, Kinney, Soper, Dodge,    Clarke-Pearson, Marks, McKenzie, Yin, Bast, “Overexpression of    HER-2/neu is associated with poor survival in advanced epithelial    ovarian cancer,” Cancer Res., 50:4087–4091, 1990.-   Berchuck, Kohler, Marks, Wiseman, Boyd, Bast, “The p53 tumor    suppressor gene frequently is altered in gynecologic cancers,”    Am. J. Obstet. Gynecol., 170:246–252, 1994.-   Berchuck, Rodriguez, Kamel, Dodge, Soper, Clarke-Pearson, Bast,    “Epidermal growth factor receptor expression in normal ovarian    epithelium and ovarian cancer. I. Correlation of receptor expression    with prognostic factors in patients with ovarian cancer,” Am. J.    Obstet. Gynecol., 164:669–674, 1991.-   Berchuck, Rodriguez, Olt, Whitaker, Boente, Arrick, Clarke-Pearson,    Bast, “Regulation of growth of normal ovarian epithelial cells and    ovarian cancer cell lines by transforming growth factor-β,” Am. J.    Obstet. Gynecol., 166:676–684, 1992.-   Berenbaum, “Synergy, additivism and antagonism in immunosuppression.    A critical review,” Clin. Exp. Immunol., 28:1–18, 1977.-   Bourne, Sanders, McCormick, “The GTPase superfamily: conserved    structure and molecular mechanism,” Nature, 349:117–127, 1991.-   Brinster et al., “Factors affecting the efficiency of introducing    foreign DNA into mice by microinjecting eggs,” Proc. Natl. Acad.    Sci. USA, 82(13):4438–4442, 1985.-   Brown, “Some properties of the Spearmann estimator in bioassay,”    Biometrika, 48:293–302, 1961.-   Brown-Shiner, Johnson, Hill, Bruskin, “Effect of protein tyrosine    phosphatase 1B expression on transformation by the human neu    oncogene,” Cancer Res., 52:478–482, 1992.-   Calvo, Vila-Jato, Alonso, “Effect of lysozyme on the stability of    polyester nanocapsules and nanoparticles: stabilization approaches,”    Biomaterials, 18(19):1305–1310, 1997.-   Calvo, Vila-Jato, Alonso, “Improved ocular bioavailability of    indomethacin by novel ocular drug carriers,” J. Pharm. Pharmacol.,    48(11):1147–1152, 1996.-   Campa, Cnang, Vedia, Reep, Lapetina, “Inhibition of Ras-induced    germinal vesicle breakdown in Xenopus oocytes by Rap-1B,” Biochem.    Biophys. Res. Commun., 174:1–5, 1991.-   Campbell, “Monoclonal Antibody Technology, Laboratory Techniques in    Biochemistry and Molecular Biology,” Vol. 13, Burden and Von    Knippenberg, Eds. pp. 75–83, Elsevier, Amsterdam, 1984.-   Capaldi et al., “Changes in order of migration of polypeptides in    complex III and cytochrome C oxidase under different conditions of    SDS polyacrylamide gel electrophoresis,” Biochem. Biophys. Res.    Commun., 74(2):425–433, 1977.-   Capaldi et al., “Isolation of a major hydrophobic protein of the    mitrochondrial inner membrane,” Biochem. Biophys. Res. Commun.,    55(3):655–659,1973.-   Capaldi, “Identification of the major enzymic activities of the    mitochondrial inner membrane in terms of their migration in sodium    dodecyl sulfate polyacrylamide gel electrophoresis,” Aech. Biochem.    Biophys., 163(1):99–105, 1974.-   Cech et al., “In vitro splicing of the ribosomal RNA precursor of    Tetrahymena: involvement of a guanosine nucleotide in the excision    of the intervening sequence,” Cell, 27(3 Pt 2):487–496, 1981.-   Chang et al., “Foreign gene delivery and expression in hepatocytes    using a hepatitis B virus vector,” Hepatology, 14:124A, 1991.-   Chen and Okayama, “High-efficiency transfection of mammalian cells    by plasmid DNA,” Mol. Cell Biol., 7:2745–2752, 1987.-   Chen et al., Nucl. Acids Res., 20:4581–4589, 1992.-   Chowrira and Burke, Nucl. Acids Res., 20:2835–2840, 1992.-   Clayman, Liu, Overholt, Mobley, Wang, Janot, Goepfert, “Gene therapy    for head and neck cancer: comparing the tumor suppressor gene p53    and a cell cycle regulator WAF1/CIP1 (p21),” Arch. Otolaryngol.,    122(5):489–93, 1996.-   Coffin, “Retroviridae and their replication,” In: Virology, Fields B    N, Knipe D M, ed., New York: Raven Press, pp. 1437–1500, 1990.-   Collins and Olive, “Reaction conditions and kinetics of    self-cleavage of a ribozyme derived from Neurospora VS RNA,”    Biochem., 32(11):2795–2799, 1993.-   Cook, Rubinfield, Albert, McCormick, “RapV12 antagonizes    Ras-dependent activation of ERK1 and ERK2 by LPA and EGF in Rat-1    fibroblasts,” EMBO J., 12:3475–3485, 1993.-   Couch et al., “Immunization with types 4 and 7 adenovirus by    selective infection of the intestinal tract,” Am. Rev. Resp. Dis.,    88:394–403, 1963.-   Coupar et al., “A general method for the construction of recombinant    vaccinia virus expressing multiple foreign genes,” Gene, Vol    68:1–10, 1988.-   Cox, “Regression Models and Life Tables” J. Royal Statistical    Society, B, 34:187–220, 1972.-   Culver et al., “In vivo gene transfer with retroviral    vector-producer cells for treatment of experimental brain tumors,”    Science, 256:1550–1552, 1992.-   Cusack, Spitz, Nguyen, Zhang, Cristiano, Roth, “High levels of gene    transduction in human lung tumors following intralesional injection    of recombinant adenovirus,” Cancer Gene Therapy, 3(4):245–249, 1996.-   Damge, Vonderscher, Marback, Pinget, “Poly(alkyl cyanoacrylate)    nanocapsules as a delivery system in the rate for octreotide, a    long-acting somatostatin analogue,” J. Pharm. Pharmacol.,    49(10):949–954, 1997.-   Dorsch, Hock, Kunzendorf, Diamantstein, Blankenstein, “Macrophage    colony-stimulating factor gene transfer into tumor cells induces    macrophage infiltration but not tumor suppression,” Eur. J.    Immunol., 23(1):186–90, 1993.-   Dropulic, Lin, Martin, Jeang, “Functional characterization of a U5    ribozyme: intracellular suppression of human immunodeficiency virus    type 1 expression,” J. Virol., 66(3):1432–41, 1992.-   Dubensky et al., “Direct transfection of viral and plasmid DNA into    the liver or spleen of mice,” Proc. Nat. Acad. Sci. USA,    81:7529–7533, 1984.-   Egli, Usman, Rich, “Conformational influence of the ribose    2′-hydroxyl group: crystal structures of DNA-RNA chimeric duplexes,”    Biochem., 32(13):3221–3237, 1993.-   El-Deiry et al., “WAF1, a potential mediator of p53 tumor    suppression,” Cell, 75:817–825, 1993.-   Elroy-Stein and Moss, “Cytoplasmic expression system based on    constitutive synthesis of bacteriophage T7 RNA polymerase in    mammalian cells,” Proc. Natl. Acad. Sci. USA, 87:6743–7, 1990.-   Fan, Baselga, Masui, Mendelsohn, “Antitumor effect of anti-epidermal    growth factor receptor monoclonal antibodies plus    cis-diamminedichloroplatinum on well established A431 cell    xenografts,” Cancer Res., 53(19):4637–42, 1993.-   Fechheimer et al., “Transfection of mammalian cells with plasmid DNA    by scrape loading and sonication loading,” Proc. Natl. Acad. Sci.    USA, 84:8463–8467, 1987.-   Feig, Bast, Knapp, Cooper, “Somatic activation of RasK gene in a    human ovarian carcinoma,” Science, 223:698–700, 1984.-   Ferkol, Lindberg, Chen, Perales, Crawford, Ratnoff, Hanson,    “Regulation of the phosphoenolpyruvate carboxykinase/human factor IX    gene introduced into the livers of adult rats by receptor-mediated    gene transfer,” FASEB J., 7(11):1081–1091, 1993.-   Ferrari, Fomasiero, Isetta, “MTT colorimetric assay for testing    macrophage cytotoxic activity in vitro,” J. Immunol. Methods,    131:165–172, 1990.-   Finney, “Statistical Methods in Biologic assays,” Third Edition. New    York Macmillan, 374–401, 1978.-   Fodor et al., “Light-directed, spatially addressable parallel    chemical synthesis,” Science, 251(4995):767–773, 1991.-   Forster and Symons, “Self-cleavage of plus and minus RNAs of a    virusoid and a structural model for the active sites,” Cell,    49:211–220, 1987.-   Fraley et al., “Entrapment of a bacterial plasmid in phospholipid    vesicles: Potential for gene transfer,” Proc. Natl. Acad. Sci. USA,    76:3348–3352, 1979.-   Frankel and Mills, “Peptide and lipid growth factors decrease    cisplatin-induced cell death in human ovarian cancer cells,” Clin.    Cancer Res., 2:1307–1313, 1996.-   Freifelder et al., “Dialysis of small samples in agarose gels,”    Anal. Biochem., 123(1):83–85, 1982.-   Freifelder et al., “Studies on Escherichia coli sex factors. I.    Specific labeling of F'Lac DNA,” J. Mol. Biol., 32(1):15–23, 1968a.-   Freifelder et al., “Studies on Escherichia coli sex factors. II.    Some physical properties of F'Lac and F DNA,” J. Mol. Biol.,    32(1):15–23, 1968b.-   Freshner, “Animal Cell Culture: a Practical Approach”, Second    Edition, Oxford/New York, IRL Press, Oxford University Press, 1992.-   Friedmann, “Progress toward human gene therapy,” Science,    244:1275–1281, 1989.-   Frohman, In: PCR™ Protocols: A Guide to Methods and Applications,    Academic Press, New York, 1990.-   Frommer, McDonald, Millar, Collis, Watt, Grigg, Molloy, Paul, “A    genomic sequencing protocol that yields a positive display of    5-methylcytosine residues in individual DNA strands,” Proc. Natl.    Acad. Sci. USA, 89(5):1827–1831, 1992.-   Gao and Huang, “Cytoplasmic expression of a reporter gene by    co-delivery of T7 RNA polymerase and T7 promoter sequence with    cationic liposomes,” Nuc. Acids Res., 21:2867–2872, 1993.-   Gefter et al., Somatic Cell Genet. 3:231–236, 1977.-   Gerlach et al., “Construction of a plant disease resistance gene    from the satellite RNA of tobacco rinspot virus,” Nature (London),    328:802–805, 1987.-   Ghosh and Bachhawat, “Targeting of liposomes to hepatocytes,” In:    Liver diseases, targeted diagnosis and therapy using specific    receptors and ligands, Wu G, Wu C ed., New York: Marcel Dekker, pp.    87–104, 1991.-   Ghosh-Choudhury, Haj-Ahmad, Graham, “Protein IX, a minor component    of the human adenovirus capsid, is essential for the packaging of    full-length genomes,” EMBO J., 6:1733–1739, 1987.-   Gibson, Leung, Squire, Hill, Arima, Goss, Hogg, Mills,    “Identification, cloning and characterization of a novel human T    cell specific tyrosine kinase located at the hematopoietin complex    on chromosome 5q,” Blood, 82:1561–1572, 1993.-   Gill, Hamel, Zhe, Zachsenhaus, Gallie, Phillips, “Characterization    of the human RB1 promoter and of elements involved in    transcriptional regulation,” Cell Growth and Differentiation,    5(5):467–474, 1994.-   Goding, “Monoclonal Antibodies: Principles and Practice,” pp. 60–74.    2nd Edition, Academic Press, Orlando, Fla., 1986.-   Gomez-Foix, Coats, Baque, Alam, Gerard, Newgard,    “Adenovirus-mediated transfer of the muscle glycogen phosphorylase    gene into hepatocytes confers altered regulation of glycogen,” J.    Biol. Chem. 267:25129–25134, 1992.-   Gopal, “Gene transfer method for transient gene expression, stable    transfection, and cotransfection of suspension cell cultures,” Mol.    Cell Biol., 5:1188–1190, 1985.-   Gossen and Bujard, “Tight control of gene expression in mammalian    cells by tetracycline responsive promoters,” Proc. Natl. Acad. Sci.    USA, 89:5547–5551, 1992.-   Graff, Herman, Lapidus, Chopra, Xu, Jarrard, Isaacs, Pitha,    Davidson, Baylin, “E-cadherin expression is silenced by DNA    hypermethylation in human breast and prostate carcinomas,” Cancer    Res., 55(22):5195–5199, 1995.-   Graham and Prevec, “Adenovirus-based expression vectors and    recombinant vaccines,” Biotechnology, 20:363–390, 1992.-   Graham and Prevec, “Manipulation of adenovirus vector,” In: Methods    in Molecular Biology: Gene Transfer and Expression Protocol, E. J.    Murray (ed.), Clifton, N.J.: Humana Press, 7:109–128, 1991.-   Graham et al., “Characteristics of a human cell line transformed by    DNA from human adenovirus type 5”, J. Gen. Virol., 36:59–72, 1977.-   Graham and van der Eb, “Transformation of rat cells by DNA of human    adenovirus 5,” Virology, 54(2):536–539, 1973.-   Grambsch and Therneau, “Proportional Hazards Tests and Diagnostics    Based On Weighted Residuals,” Biometrika, 81:5515–5526, 1994.-   Grunhaus and Horwitz, “Adenovirus as cloning vector,” Seminar in    Virology, 3:237–252, 1992.-   Guerrier-Takada, Gardiner, Marsh, pace, Altman, “The RNA moiety of    ribonuclease P is the catalytic subunit of the enzyme,” Cell,    35:849, 1983.-   Gum, Lengyel, Juarez, Chen, Sato, Seiki, Boyd, “Stimulation of    92-kDa gelatinase B promoter activity by ras is mitogen-activated    protein kinase 1-independent and requires multiple transcription    factor binding sites including closely spaced PEA3/ets and AP-1    sequences,” J. Biol. Chem., 271(18):10672–10680, 1996.-   Hagopian, Mills, Khokhar, Bast, Siddik, “Studies of cisplatin (CDDP)    resistance with 1R,2R-diaminocyclohexane    (DACH)-diacetato-dichloro-Pt(IV) (acetato-Pt) in ovarian cancer cell    lines,” Proc. Amer. Assoc. Cancer Res., 37:402(A#1399), 1996.-   Hampel and Tritz, “RNA catalytic properties of the minimum (−)s TRSV    sequence,” Biochem., 28:4929, 1989.-   Hampel, Tritz, Hicks, Cruz, “‘Hairpin’ catalytic RNA model: evidence    for helices and sequence requirement for substrate RNA,” Nuc. Acids    Res., 18:299, 1990.-   Harland and Weintraub, “Translation of mammalian mRNA injected into    Xenopus oocytes is specifically inhibited by antisense RNA,” J. Cell    Biol., 101:1094–1099, 1985.-   Harlow and Lane, “Antibodies: A Laboratory Manual,” Cold Spring    Harbor Laboratory, Cold Spring Harbor, N.Y., 1988.-   Harrington and Fleming, “Counting Processes and Survival Analysis,”    John Wiley and Sons, New York, 1991.-   Hata, Kaibuchi, Kawamura, Hiroyoshi, Shirataki, Takai, “Enhancement    of the actions of smg p21 GDP/GTP exchange protein by the protein    kinase A-catalyzed phosphorylation of smg p21,” J. Biol. Chem.,    166:6571–6577, 1991.-   Havrilesky, Hurteau, Whitaker, Elbendary, Wu, Rodriguez, Bast,    Berchuck, “Regulation of apoptosis in normal and malignant ovarian    epithelial cells by transforming growth factor-β” Cancer Res.,    55:944–948, 1995.-   Herman, Jen, Merlo, Baylin, “Hypermethylation-associated    inactivation indicates a tumor suppressor role for p15INK4B,” Cancer    Res., 56(4):722–727, 1996.-   Herman, Latif, Weng, Lerman, Zbar, Liu, Samid, Duan, Gnarra, Linehan    et al., “Silencing of the VHL tumor-suppressor gene by DNA    methylation in renal carcinoma,” Proc. Natl. Acad. Sci. USA,    91(21):9700–9704, 1994.-   Hermonat and Muzycska, “Use of adenoassociated virus as a mammalian    DNA cloning vector: Transduction of neomycin resistance into    mammalian tissue culture cells,” Proc. Natl. Acad. Sci. USA,    81:6466–6470, 1984.-   Hersdorffer et al., “Efficient gene transfer in live mice using a    unique retroviral packaging line,” DNA Cell Biol., 9:713–723, 1990.-   Hertel, Pardi, Uhlenbeck, Koizumi, Ohtsuka, Uesugi, Cedergren,    Eckstein, Gerlach, Hodgson et al., “Numbering system for the    hammerhead,” Nucl. Acids Res., 20(12):3252, 1992.-   Herz and Gerard, “Adenovirus-mediated transfer of low density    lipoprotein receptor gene acutely accelerates cholesterol clearance    in normal mice,” Proc. Natl. Acad. Sci. USA, 90:2812–2816, 1993.-   Hilsenbeck, and Clark, “Practical p-Value Adjustment for Optimally    Selected Cutpoints,” Statistics in Medicine, 15:103–112, 1996.-   Hoggard, Brintnell, Howell, Weissenbach, Varley, “Allelic imbalance    on chromosome 1 in human breast cancer. II. Microsatellite repeat    analysis,” Genes, Chromosomes Cancer, 12:24–31, 1995.-   Horwich et al., “Synthesis of hepadnavirus particles that contain    replication-defective duck hepatitis B virus genomes in cultured    HuH7 cells,” J. Virol., 64:642–650, 1990.-   Hurteau, Rodriguez, Whitaker, Shah, Mills, Bast, Berchuck,    “Transforming growth factor-β inhibits proliferation of human    ovarian cancer cells obtained from ascites,” Cancer, 74:93–99, 1994.-   Innis et al., “DNA sequencing with Thermus aquaticus DNA polymerase    and direct sequencing of polymerase chain reaction-amplified DNA,”    Proc. Natl. Acad. Sci. USA, 85(24):9436–9440, 1988.-   Jacobs, Kohler, Wiseman, Marks, Whitaker, Kerns, Humphrey, Berchuck,    Ponder, Bast, “Clonal origin of epithelial ovarian cancer: Analysis    by loss of heterozygosity, p53 mutation and X chromosome    inactivation,” J. Natl. Cancer Inst., 84:1793–1798, 1992.-   Jacobs, Smith, Wiseman, Futreal, Harrington, Osborne, Leach,    Molyneaux, Berchuck, Ponder, Bast, “A deletion unit on chromosome    17q in epithelial ovarian tumors distal to the familial    breast/ovarian cancer locus,” Cancer Res., 53:1218–1221, 1993.-   Jaeger, Turner, Zuker, “Improved predictions of secondary structures    for RNA,” Proc. Natl. Acad. Sci. USA, 86(20):7706–7710, 1989.-   Jahnke, Van de Stolpe, Caldenhoven, Johnson, “Constitutive    expression of human intercellular adhesion molecule-1 (ICAM-1) is    regulated by differentially active enhancing and silencing elements,    Eur. J. Biochem, 228(2):439–446, 1995.-   Jameson and Wolf, “The antigenic index: a novel algorithm for    predicting antigenic determinants,” Compu. Appl. Biosci.,    4(1):181–186, 1988.-   Jelinek and Hassell, “Reversion of middle T antigen-transformed    Rat-2 cells by Krev-1: implications for the role of p21c-Ras in    polyomavirus-mediated transformation,” Oncogene, 7:1687–1698, 1992.-   Jones and Shenk, “Isolation of deletion and substitution mutants of    adenovirus type 5,” Cell, 13:181–188, 1978.-   Joyce, “RNA evolution and the origins of life,” Nature, 338:217–244,    1989.-   Kacinski, “CSF-1 and its receptor in ovarian, endometrial and breast    cancer,” Ann. Med, 27(1):79–85, 1995.-   Kacinski, Mayer, King et al., “Neu protein overexpression in benign,    borderline, and malignant ovarian neoplasms,” Gynecol. Oncol.,    44:245–253, 1992.-   Kaneda et al., “Increased expression of DNA cointroduced with    nuclear protein in adult rat liver,” Science, 243:375–378, 1989.-   Karlan, Baldwin, Cirisano, Mamula, Jones, Lagasse, “Secreted ovarian    stromal substance inhibits ovarian epithelial cell proliferation,”    Gyn. Onc, 59(1):67–74, 1995.-   Kashani-Saber et al., Antisense Res. Dev., 2:3–15, 1992.-   Kato et al., “Expression of hepatitis B virus surface antigen in    adult rat liver,” J. Biol. Chem., 266:3361–3364, 1991.-   Kitayama, Sugimoto, Matsuzaki, Ikawa, Noda, “A Ras-related gene with    transformation suppressor activity,” Cell, 56:77–84, 1989.-   Klein et al., Proc. Natl. Acad. Sci. USA, 85:8502–8505, 1988.-   Klein, Wolf, Wu, Sanford, “High-velocity microprojectiles for    delivering nucleic acids into living cells,” Nature, 327:70–73,    1987.-   Kohler and Milstein, “Derivation of specific antibody-producing    tissue culture and tumor lines by cell fusion,” Eur. J. Immunol.,    6(7):511–519, 1976.-   Kohler and Milstein, “Continuous cultures of fused cells secreting    antibody of predefined specificity,” Nature, 256(5517):495–497,    1975.-   Kohler, Marks, Wiseman, Jacobs, Davidoff, Clarke-Pearson, Soper,    Bast, Berchuck, “Spectrum of mutation and frequency of allelic    deletion of the p53 gene in ovarian cancer,” J. Natl. Cancer Inst.,    85:1513–1519, 1993.-   Korn, and Simon, “Measures of explained variation for survival    data,” Statistics in Medicine, 9:487–503, 1990.-   Kornblau, Thall, Yang, Estey, Andreeff, “Analysis of CD7 Expression    in acute myelogenous leukemia: Martingale residual plots combined    with ‘optimal’ cutpoint analysis reveals absence of prognostic    significance,” Leukemia, 9:1735–1741, 1995.-   Kroning, Jones, Hom, Chuang, Sanga, Los, Howell, Christen,    “Enhancement of drug sensitivity of human malignancies by epidermal    growth factor,” Brit. J. Cancer, 72(3):615–619, 1995.-   Kruk, Maines-Bandiera, Auersperg, “A simplified method to culture    human ovarian surface epithelium,” Lab. Invest., 63(1):132–136,    1990.-   Kuby, “Immunology” 2nd Edition. W.H. Freeman & Company, New York,    1994.-   Kunkel, Roberts, Zakour, “Rapid and efficient site-specific    mutagenesis without phenotypic selection,” Methods Enzymol.,    154:367–382, 1987.-   Kwoh et al., Proc. Natl. Acad. Sci, USA, 86(4):1173–1177, 1989.-   Kyte and Doolittle, J. Mol. Biol., 157:105–132, 1982.-   L'Huillier, David, Bellamy, “Cytoplasmic delivery of ribozymes leads    to efficient reduction in alpha-lactalbumin mRNA levels in C1271    mouse cells,” EMBO J., 11(12):4411–4418, 1992.-   Le Gal La Salle, Robert, Berrard, Ridoux, Stratford-Perricaudet,    Perricaudet, Mallet, “An adenovirus vector for gene transfer into    neurons and glia in the brain,” Science, 259:988–990, 1993.-   Lee et al., “Human retinoblastoma susceptibility gene: cloning,    identification, and sequence,” Science, 235:1394–1399, 1987.-   Levrero, Barban, Manteca, Ballay, Balsamo, Avantaggiati, Natoli,    Skellekens, Tiollais, Perricaudet, “Defective and nondefective    adenovirus vectors for expressing foreign genes in vitro and in    vivo,” Gene, 101:195–202, 1991.-   Li, Han, Resnik, Carcangiu, Schwartz, Yang-Feng, “Advanced ovarian    carcinoma: molecular evidence of unifocal origin,” Gyn. Onc.,    51(1):21–5, 1993.-   Liang and Pardee, “Differential display of eukaryotic messenger RNA    by means of the polymerase chain reaction,” Science, 257:967–971,    1992.-   Lidor, Shpall, Peters, Bast, “Synergistic cytotoxicity of different    alkylating agents for epithelial ovarian cancer,” Int. J. Cancer,    49(5):704–710, 1991.-   Lidor, Xu, Martinez-Maza, Olt, Marks, Berchuck, Ramakrishnan, Berek,    Bast, “Constitutive production of macrophage colony stimulating    factor and interleukin-6 by human ovarian surface epithelial cells,”    Exp. Cell Res., 207:332–339, 1993.-   Lieber, Sandig, Sommer, Bahring, Strauss, “Stable high-level gene    expression in mammalian cells by T7 phage RNA polymerase,” Methods    Enzymol., 217:47–66, 1993.-   Lin, “Goodness-of-fit analysis for the Cox regression model based on    a class of parameter estimators,” J. American Statistical    Association, 86:725–728. 1991.-   Lisziewicz et al., Proc. Natl. Acad. Sci. USA, 90:8000–8004, 1993.-   Lounis et al., “Primary cultures of normal and tumoral human ovarian    epithelium: a powerful tool for basic molecular studies,” Exp. Cell    Res., 215:303–309, 1994.-   Loupart, Armour, Walker, Adams, Brammar, Varley, “Allelic imbalance    on chromosome 1 in human breast cancer. I. Ministellite and RFLP    analysis,” Genes Chromosomes Cancer, 12:16–23, 1995.-   Lowe and Temple, “Calcitonin and insulin in isobutylcyanoacrylate    nanocapsules: protection against proteases and effect on intestinal    absorption in rats,” 46(7):547–552, 1994.-   Lynch, Smyrk, Lynch, “Overview of natural history, pathology,    molecular genetics and management of HNPCC (Lynch Syndrome),”    Int. J. Cancer, 69(1):38–43, 1996.-   Malkin, Li, Strong et al., “Germ line p53 mutations in a familial    syndrome of breast cancer, sarcomas, and other neoplasms,” Science,    250:1233–1238, 1990.-   Maloy et al., “Microbial Genetics” 2nd Edition. Jones and Barlett    Publishers, Boston, Mass., 1994.-   Mann et al., “Construction of a retrovirus packaging mutant and its    use to produce helper-free defective retrovirus,” Cell, 33:153–159,    1983.-   Markowitz et al., “A safe packaging line for gene transfer:    Separating viral genes on two different plasmids,” J. Virol.,    62:1120–1124, 1988.-   Merrifield B., “Solid phase synthesis,” Science, 232(4748):341–347,    1986.-   Michael, Biotechniques, 16:410–412, 1994.-   Michel and Westhof, “Modeling of the three-dimensional architecture    of group I catalytic introns based on comparative sequence    analysis,” J. Mol. Biol., 216:585–610, 1990.-   Michieli et al., “Induction of WAF1/CIP1 by a p53-independent    pathway,” Cancer Res., 54:3391–3395, 1994.-   Miki, Swensen, Shattuck-Eidens et al., “A strong candidate for the    breast and ovarian cancer susceptibility gene BRCA1,” Science,    266:66–71, 1994.-   Miura, Kaibuchi, Itoh, Corbin, Francis, Takai, “Phosphorylation of    smg p21B/Rap1B p21 by cyclic GMP-dependent protein kinase,” FEB.    Lett., 297:171–174, 1992.-   Mok, Tsao, Knapp, Fishbaugh, Lau, “Unifocal origin of advanced human    epithelial ovarian cancers,” Cancer Res., 52:5119–5122, 1992.-   Morishige, Kurachi, Amemiya, Adachi, Inoue, Miyake, Tanizawa,    Sakoyama, “Involvement of transforming growth factor alpha/epidermal    growth factor receptor autocrine growth mechanism in an ovarian    cancer cell line in vitro,” Cancer Res., 51(21):5951–5955, 1991.-   Moser, Young, Rodriguez, Pizzo, Bast, Stack, “Secretion of    extracellular matrix-degrading proteinases is increased in    epithelial ovarian carcinomas,” Int J. Cancer, 56:552–559, 1994.-   Mujoo, Maneval, Anderson, Gutterman, “Adenoviral-mediated p53 tumor    suppressor gene therapy of human ovarian carcinoma,” Oncogene,    12(8):1617–1623, 1996.-   Nagai, Negrini, Carter, Gillum, Rosenberg, Schwartz, Croce,    “Detection and cloning of a common region of loss of heterozygosity    at chromosome 1p in breast cancer,” Cancer Res., 55:1752–1757, 1995.-   Nicolas and Rubenstein, “Retroviral vectors,” In: Vectors: A survey    of molecular cloning vectors and their uses, Rodriguez R L, Denhardt    D T, ed., Stoneham: Butterworth, pp. 493–513, 1988.-   Nicolau and Sene, “Liposome-mediated DNA transfer in eukaryotic    cells,” Biochim. Biophys. Acta, 721:185–190, 1982.-   Nicolau et al., “Liposomes as carriers for in vivo gene transfer and    expression,” Methods Enzymol., 149:157–176, 1987.-   Ohara et al., Proc. Natl. Acad. Sci. USA, 86(15):5673–5677, 1989.-   Ohara, Dorit, Gilbert, “Direct genomic sequencing of bacterial DNA:    the pyruvate kinase I gene of Escherichia coli,” Proc. Natl. Acad.    Sci. USA, 86(18):6883–6887, 1989.-   Ohkawa, Yuyama, Taira, “Activities of HIV-RNA targeted ribozymes    transcribed from a ‘shot-gun’ type ribozyme-trimming plasmid,” Nucl.    Acids Symp. Ser., 27:15–6, 1992.-   Ohtani-Fujita, ujita, Aoike, Osifchin, Robbins, Sakai, “CpG    methylation inactivates the promoter activity of the human    retinoblastoma tumor-suppressor gene,” Oncogene, 8(4):1063–1067,    1993-   Ojwang, Hampel, Looney, Wong-Staal, Rappaport, “Inhibition of human    immunodeficiency virus type 1 expression by a hairpin ribozyme,”    Proc. Natl. Acad. Sci. USA., 89(22):10802–10806, 1992.-   Paskind et al., “Dependence of moloney murine leukemia virus    production on cell growth,” Virology, 67:242–248, 1975.-   Patton, Jameson, Martin, Altschuler, Bast, Ostrowski, “Activated ras    signaling and uPA expression in ovarian carcinoma,” Fifth Meeting on    the Molecular Basis of Cancer, Hood College, Frederick, Md., 1994.-   Pease et al., Light-generated oligonucleotide arrays for rapid DNA    sequence analysis,” Proc. Natl. Acad. Sci. USA, 91(11):5022–5026,    1994.-   Pelletier and Sonenberg, “Internal initiation of translation of    eukaryotic mRNA directed by a sequence derived from poliovirus RNA,”    Nature, 334(6180):320–325, 1988.-   Perales, Ferkol, Beegen, Ratnoff, Hanson, “Gene transfer in vivo:    sustained expression and regulation of genes introduced into the    liver by receptor-targeted uptake,” Proc. Natl. Acad. USA,    91(9):4086–4090, 1994.-   Perreault, Wu, Cousinequ, Ogilvie, Cedergren, “Mixed deoxyribo- and    ribo-oligonucleotides with catalytic activity,” Nature,    344(6266):565, 1990.-   Perrotta and Been, “Cleavage of oligoribonucleotides by a ribozyme    derived from the hepatitis delta virus RNA sequence,” Biochem.,    31(1):16, 1992.-   Pieken, Olsen, Benseler, Aurup, Eckstein, “Kinetic characterization    of ribonuclease-resistant 2′-modified hammerhead ribozymes,”    Science, 253(5017):314, 1991.-   Pignon et al., “Exhaustive analysis of the P53 gene coding sequence    by denaturing gradient gel electrophoresis: application to the    detection of point mutations in acute leukemias,” Hum. Mutat.,    3(2):126–132, 1994.-   Pizon, Chardin, Lerosey, Olofsson, Tavitian, “Human cDNAs Rap1 and    Rap2 homologous to the Drosophila gene DRas3 encode proteins closely    related to Ras in the ‘effector’ region,” Oncogene, 3:210–204, 1988.-   Potter et al., “Enhancer-dependent expression of human k    immunoglobulin genes introduced into mouse pre-B lymphocytes by    electroporation,” Proc. Nat. Acad. Sci. USA, 81:7161–7165, 1984.-   Pregibon, “Resistant fits for some commonly used logistic models    with medical applications,” Biometrics, 38:485–498, 1982.-   Prokop and Bajpai, Ann. N.Y. Acad. Sci., Vol. 646, 1991.-   Quilliam, Mueller, Bohl, Prossnitz, Sklar, Der, Bokoch, “Rap1A is a    substrate for cyclic AMP-dependent protein kinase in human    neutrophils,” J. Immunol., 147:1628–1635, 1991.-   Ragot, Vincent, Chafey, Vigne, Gilgenkrantz, Couton, Cartaud,    Briand, Kaplan, Perricaudet, Kahn, “Efficient adenovirus-mediated    transfer of a human minidystrophin gene to skeletal muscle of mdx    mice,” Nature 361:647–650, 1993.-   Reinhold-Hurek and Shub, “Self-splicing introns in tRNA genes of    widely divergent bacteria,” Nature, 357:173–176, 1992.-   Rich et al., “Development and analysis of recombinant adenoviruses    for gene therapy of cystic fibrosis,” Hum. Gene Ther., 4(4):461–476,    1993.-   Ridgeway, “Mammalian expression vectors,” In: Vectors: A survey of    molecular cloning vectors and their uses, Rodriguez R L, Denhardt D    T, ed., Stoneham: Butterworth, pp. 467–492, 1988.-   Rippe et al., “DNA-mediated gene transfer into adult rat hepatocytes    in primary culture,” Mol. Cell Biol., 10:689–695, 1990.-   Rodriguez, Berchuck, Whitaker, Schlossman, Clarke-Pearson, Bast,    “Epidermal growth factor receptor expression in normal ovarian    epithelium and ovarian cancer. II. Relationship between receptor    expression and response to epidermal growth factor” Am. J. Obstet.    Gynecol., 164:745–750, 1991.-   Rosenfeld, Yoshimura, Trapnell, Yoneyama, Rosenthal, Dalemans,    Fukayama, Bargon, Stier, Stratford-Perricaudet, Perricaudet,    Guggino, Pavirani, Lecocq, Crystal, “In vivo transfer of the human    cystic fibrosis transmembrane conductance regulator gene to the    airway epithelium,” Cell, 68:143–155, 1992.-   Rosenfeld, Siegfried, Yoshimura, Yoneyama, Fukayama, Stier, Paakko,    Gilardi, Stratford-Perricaudet, Perricaudet, Jallat, Pavirani,    Lecocq, Crystal, “Adenovirus-mediated transfer of a recombinant    ∀1-antitrypsin gene to the lung epithelium in vivo,” Science,    252:431–434, 1991.-   Rossi, Elkins, Zaia, Sullivan, “Ribozymes as anti-HIV-1 therapeutic    agents: principles, applications, and problems,” AIDS Res. Hum.    Retrovir., 8(2): 183, 1992.-   Roux et al., “A versatile and potentially general approach to the    targeting of specific cell types by retroviruses: Application to the    infection of human cells by means of major histocompatibility    complex class I and class II antigens by mouse ecotropic murine    leukemia virus-derived viruses,” Proc. Natl. Acad. Sci. USA,    86:9079–9083, 1989.-   Rubin, Finstad, Wong et al, “Prognostic significance of HER-2/neu    expression in advanced ovarian cancer,” Am. J. Obstet. Gynecol.,    168:162–169, 1993.-   Sahyoun, McDonald, Farrell, Lapetina, “Phosphorylation of a    Ras-related GTP-binding protein, Rap-1b, by a neuronal    Ca2+/calmodulin-dependent protein kinase, CaM kinase Gr,” Proc.    Natl. Acad. Sci. USA, 88:2643–2647, 1991.-   Sakoda, Kaibuchi, Kishi, Kishida, Doi, Hoshino, Hattori, Takai,    “smg/Rap1/Krev-1/p21s inhibit the signal pathway to the c-fos    promoter/enhancer from c-Ki-Ras p21 but not from c-raf-1 kinase in    NIH3T3 cells,” Oncogene, 7:1705–1711, 1992.-   Sambrook et al., “Molecular Cloning: A Laboratory Manual,” Cold    Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989.-   Sarver, Cantin, Chang, Zaia, Ladne, Stephens, Rossi, “Ribozymes as a    potential anti-HIV-1 therapeutic agents,” Science,    247(4947):1222–1225, 1990.-   Saville and Collins, “A site-specific self-cleavage reaction    performed by a novel RNA in Neurospora mitochondria,” Cell,    61(4):685–696, 1990.-   Saville and Collins, “RNA-mediated ligation of self-cleavage    products of a Neurospora mitochondrial plasmid transcript,” Proc.    Natl. Acad. Sci. USA, 88(19):8826–8830, 1991.-   Scanlon, Jiao, Funato, Wang, Tone, Rossi, Kashani-Sabet,    “Ribozyme-mediated cleavage of c-fos mRNA reduces gene expression of    DNA synthesis enzymes and metallothionein,”, Proc. Natl. Acad. Sci.    USA, 88(23):10591–10595, 1991.-   Scaringe, Francklyn, Usman, “Chemical synthesis of biologically    active oligoribonucleotides using beta-cyanoethyl protected    ribonucleoside phosphoramidites,” Nucl. Acids Res.,    18(18):5433–5441, 1990.-   Schemper and Stare, “Explained Variation in Survival Analysis,”    Statistics in Medicine, 15:1999–2012, 1996.-   Segal, “Biochemical Calculations,” 2nd Edition, John Wiley & Sons,    New York, 1976.-   Shultz, Schwaitzer, Rajan, Yi, Ihle, Mathews, Thomas, Beier,    “Mutations at the murine motheaten locus are within the    hematopoietic cell protein-tyrosine phosphatase (Hcph) gene,” Cell,    73(7):1445–54, 1993.-   Stampfer, “Isolation and growth of human mammary epithelial    cells,” J. Tissue Culture Methods, 9:107–115, 1985.-   Steel and Peckham, “Exploitable mechanisms in combined    ratiotherapy-chemotherapy: The concept of additivity,” Int. J.    Radiation Oncol. Biol. Phys., 5:85–91, 1979.-   Stewart et al., “Immunochemical studies on tobacco mosaic virus    protein. IV. The automated solid-phase synthesis of a decapeptide of    tobacco mosaic virus protein and its reaction with antibodies to the    whole protein,” Biochemistry, 5(11):3396–3400, 1966.-   Stratford-Perricaudet and Perricaudet, “Gene transfer into animals:    the promise of adenovirus,” p. 51–61, In: Human Gene Transfer,    Eds, O. Cohen-Haguenauer and M. Boiron, Editions John Libbey    Eurotext, France, 1991.-   Stratford-Perricaudet et al., “Evaluation of the transfer and    expression in mice of an enzyme-encoding gene using a human    adenovirus vector,” Hum. Gene Ther., 1:241–256, 1990.-   Stromberg, Collins, Gordon, Jackson, Johnson, “Transforming growth    factor-alpha acts as an autocrine growth factor in ovarian carcinoma    cell lines,” Cancer Res., 52(2):341–347, 1992.-   Symmans, Liu, Knowles, Inghirami, “Breast cancer heterogeneity:    evaluation of clonality in primary and metastatic lesions,” Hum.    Path., 26:210–216, 1995.-   Taira, Nakagawa, Nishikawa, Furukawa, “Construction of a novel    RNA-transcript-trimming plasmid which can be used both in vitro in    place of run-off and (G)-free transcriptions and in vivo as    multi-sequences transcription vectors,” Nucl. Acids Res.,    19(19):5125–5130, 1991.-   Temin, “Retrovirus vectors for gene transfer: Efficient integration    into and expression of exogenous DNA in vertebrate cell genome,” In:    Gene transfer, Kucherlapati R, ed., New York: Plenum Press, pp.    149–188, 1986.-   Therneau, “A Package for Survival Analysis in S,” Mayo Foundation,    1994.-   Therneau, Grambsch, Fleming, “Martingale-based Residuals for    Survival Models,” Biometrika, 77:147–160, 1990.-   Tomic, Sunjevaric, Savtchenko, Blumenberg, “A rapid and simple    method for introducing specific mutations into any position of DNA    leaving all other positions unaltered,” Nucl. Acids Res.,    18(6):1656, 1990.-   Top et al., “Immunization with live types 7 and 4 adenovirus    vaccines. II. Antibody response and protective effect against acute    respiratory disease due to adenovirus type 7,” J. Infect. Dis.,    124:155–160, 1971.-   Tornaletti and Pfeifer, “Complete and tissue-independent methylation    of CpG sites in the p53 gene: implications for mutations in human    cancers,” Oncogene 10(8):1493–1499, 1995.-   Tur-Kaspa et al., “Use of electroporation to introduce biologically    active foreign genes into primary rat hepatocytes,” Mol. Cell Biol.,    6(2):716–718, 1986.-   Upender, Raj, Weir, “Megaprimer method for in vitro mutagenesis    using parallel templates,” Biotechniques, 18:29–31, 1995.-   Usman and Cedergren, “Exploiting the chemical synthesis of RNA,”    Trends in Biochem. Sci., 17(9):334, 1992.-   Usman et al., J. Am. Chem. Soc., 109:7845–7854, 1987.-   Varmus et al., “Retroviruses as mutagens: Insertion and excision of    a nontransforming provirus alter the expression of a resident    transforming provirus,” Cell, 25:23–36, 1981.-   Venables, Ripley, Springer-Verlag, “Modern Applied Statistics With    S-Plus,” New York, 1994.-   Ventura, Wang, Ragot, Perricaudet, Saragosti, Nucl. Acids Res.,    21(14):3249–3255, 1993.-   Voss et al., “Synthesis of the protected tridecapeptide (56–68) of    the VH domain of mouse myeloma immunoglobulin M603 and its    reattachment to resin supports,” Int J. Pept. Protein Res.,    22(2):204–213, 1983.-   Wagner, Zenke, Cotten, Beug, Birnstiel, “Transferrin-polycation    conjugates as carriers for DNA uptake into cells,” Proc. Natl. Acad.    Sci. USA, 87:3410–3414, 1990.-   Walker, Little, Nadeau, Shank, “Isothermal in vitro amplification of    DNA by a restriction enzyme/DNA polymerase system,” Proc. Natl.    Acad. Sci. USA, 89(1):392–396, 1992.-   Watson, et al., Molecular Biology of the Gene, 4th Ed., W. A.    Benjamin, Inc., Menlo Park, Calif., 1987.-   Weerasinghe, Liem, Asad, Read, Joshi, “Resistance to human    immunodeficiency virus type 1 (HIV-1) infection in human CD4+    lymphocyte-derived cell lines conferred by using retroviral vectors    expression an HIV-1 RNA-specific ribozyme,” J. Virol.,    65(10):5531–5534, 1991.-   Weinberg, “Positive and negative controls on cell growth,”    Biochemistry, 28:8263–8269, 1989.-   Wiener, Hurteau, Kems, Whitaker, Conaway, Wu, Berchuck, Bast,    “Overexpression of the tyrosine phosphatase PTP1B is associated with    human ovarian carcinomas,” Am. J. Obstet. Gynecol., 170:1177–1183,    1994.-   Wiener, Kassim, Yu, Mills, Bast, “Transfection of human ovarian    cancer cells with the HER-2/neu receptor tyrosine kinase induces a    selective increase in PTP-H1, PTP-1B, and PTP-expression,” Gynecol.    Oncol., 61:223–240, 1996.-   Wolf et al., “An Integrated Family of Amino Acid Sequence Analysis    Programs,” Compu. Appl. Biosci., 4(1):187–191, 1988.-   Wolf, Bazelle, Mills, Bast, Roth, Gershenson, “Growth inhibition of    human ovarian cancer cells by transfection with adenovirus-mediated    p53 is independent of endogenous p53 status,” Proc. Amer. Assoc.    Cancer Res., 37:205(A#1399), 1996.-   Wong et al., “Appearance of β-lactamase activity in animal cells    upon liposome mediated gene transfer,” Gene, 10:87–94, 1980.-   Woolf, Melton, Jennings, Proc. Natl. Acad. Sci. USA,    89(16):7305–7309, 1992.-   Wooster, Neuhausen, Mangion et al., “Localization of a breast cancer    susceptibility gene, BRCA2, to chromosome 13q12–13,” Science,    265:2088–2090, 1994.-   Worsley, Ponder, Davies, “Overexpression of cyclin D1 in epithelial    ovarian cancers,” Gynecol. Oncol., 64:189–195, 1997.-   Wu and Wang, “Sequence-selective DNA binding to the regulatory    subunit of cAMP-dependent protein kinase,” J. Biol. Chem.,    264(17):9989–9993, 1989.-   Wu and Wu, “Evidence for targeted gene delivery to HepG2 hepatoma    cells in vitro,” Biochemistry, 27:887–892, 1988.-   Wu and Wu, “Receptor-mediated in vitro gene transfections by a    soluble DNA carrier system,” J. Biol. Chem., 262:4429–4432, 1987.-   Wu and Wu, Adv. Drug Delivery Rev., 12:159–167, 1993.-   Wu, Rodabaugh, Martinez-Maza, Watson, Silberstein, Boyer, Peters,    Weinberg, Berek, Bast, “Stimulation of ovarian tumor cell    proliferation with monocyte products including interleukin-1,    interleukin-6, and tumor necrosis factor-alpha,” Am. J. Obstet.    Gynecol., 166:997–1007, 1992.-   Wu and Dean, “Functional significance of loops in the receptor    binding domain of Bacillus thuringiensis CrylIIA δ-endotoxin,” J.    Mol. Biol., 255(4):628–640, 1996.-   Xiong et al., “p21 is a universal inhibitor of cyclin kinases,”    Nature, 366:701–704, 1993.-   Xu et al., “Development of two new monoclonal antibodies reactive to    a surface antigen present on human ovarian epithelial cancer cells,”    Cancer Res., 51:4012–4019, 1991.-   Xu, Fang, Gaudette, Holub, Casey, Mills, “Lysophospholipids activate    ovarian and breast cancer cells,” Biochem. J, 309:933–940. 1995.-   Xu, Ramakrishnan, Daly, Soper, Berchuck, Clarke-Pearson, Bast,    “Increased serum levels of macrophage colony-stimulating factor in    ovarian cancer,” Am. J. Obstet. Gynecol., 165:1356–1362, 1991.-   Xu, Rodriguez, Bae, Whitaker, Boyer, Mills, Yu, Bast, “Heregulin and    anti-p185c-erb^(B-2) antibodies inhibit proliferation, increase    invasiveness and enhance tyrosine autophosphorylation of breast    cancer cells that overexpress p185c-erb^(B-2) ,” Proc. Amer. Assoc.    Cancer Res., 35:38(A#225), 1994.-   Xu, Yu, Boyer, Walch, Khan, Mills, Bast, “Stimulation or inhibition    of ovarian cancer cell proliferation by heregulin is dependent on    the ratio of HER2 to HER3 or HER4 expression,” Proc. Amer. Assoc.    Cancer Res., 37:191(A#1305), 1996.-   Yang et al., “In vivo and in vitro gene transfer to mammalian    somatic cells by particle bombardment,” Proc. Natl. Acad. Sci. USA,    87:9568–9572, 1990.-   Yatani, Quilliam, Brown, Bokoch, “Rap1A antagonizes the ability of    Ras and Ras-Gap to inhibit muscarinic K+ channels,” J. Biol. Chem.,    266:22222–22226, 1991.-   Young, Rodriguez, Moser, Bast, Pizzo, Stack, “Coordinate expression    of urinary-type plasminogen activator and its receptor accompanies    malignant transformation of the ovarian surface epithelium,” Am. J.    Obstet. Gynecol., 170:1285–1296, 1994.-   Young, Rodriguez, Rinehart, Bast, Pizzo, Stack, “Characterization of    gelatinases linked to extracellular matrix invasion in ovarian    adenocarcinoma: purification of matrix metalloproteinase 2,” Gyn.    Oncol., 62:89–99, 1996.-   Yu and Chang, “Submicron polymer membrane hemoglobin nanocapsules as    potential blood substitutes: preparation and characterization,”    Artif Cells Blood Substit. Immobil. Biotechnol., 24(3): 169–183,    1996.-   Yu, Ojwang, Yamada, Hampel, Rapapport, Looney, Wong-Staal, “A    hairpin ribozyme inhibits expression of diverse strains of human    immunodeficiency virus type 1,” Proc. Natl. Acad. Sci. USA,    90:6340–6344, 1993.-   Yu, Henry, Xu, Hamilton, “Expression of a murine cytomegalovirus    early and late protein in latently infected mice,” J. Infectious    Diseases, 172:371–379, 1995a.-   Yu, Matin, Xia, Sorgi, Huang, Hung, “Liposome-mediated in vivo E1A    gene transfer suppressed dissemination of ovarian cancer cells that    overexpress HER-2/neu,” Oncogene, 11(7):1383–1388, 1995b.-   Zelenin et al., “High-velocity mechanical DNA transfer of the    chloramphenicol acetyltransferase gene into rodent liver, kidney and    mammary gland cells in organ explants and in vivo,” FEBS Lett.,    280:94–96, 1991.-   Zhang, Calaf, Russo, “Allele loss and point mutation in codons 12    and 61 of the c-Ha-ras oncogene in carcinogen-transformed human    breast epithelial cells,” Mol. Carcin., 9:46–56, 1994.-   Zhou, Giordano, Durbin, McAllister, Mol. Cell Biol.,    10(9):4529–4537, 1990.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

1. An isolated or purified polynucleotide that encodes the amino acidsequence of SEQ ID NO:2, or the complete complement of such apolynucleotide.
 2. The polynucleotide of claim 1, further defined ascomprising the nucleic acid sequence of from position 150 to position833 of SEQ ID NO:1 or the nucleic acid sequence of SEQ ID NO:5.
 3. Thepolynucleotide of claim 1, further defined as a sequence complementaryto the nucleic acid sequence of from position 150 to position 833 of SEQID NO:1 or the nucleic acid sequence of SEQ ID NO:5.
 4. Thepolynucleotide of claim 1, wherein said polynucleotide is operablylinked to a promoter.
 5. The polynucleotide of claim 1, comprised withina vector.
 6. The polynucleotide of claim 5, wherein said vector is aplasmid, cosmid, phagemid, virus, baculovirus, yeast artificialchromosome, bacterial artificial chromosome or phage.
 7. Thepolynucleotide of claim 6, wherein said virus is an adenovirus, anadenoassociated virus, a retrovirus, a Herpes virus, or a vacciniavirus.
 8. A host cell comprising the polynucleotide selected from thegroup consisting of the polynucleotides of any one of claims 1–3 and4–7.
 9. The host cell of claim 8, further defined as a bacterial cell.10. The host cell of claim 9, wherein said bacterial cell is an E. coli,Pseudomonas sp. or salmonella cell.
 11. The host cell of claim 8,further defined as a eukaryotic cell.
 12. The host cell of claim 11,further defined as an animal cell, a yeast cell, or a fungal cell. 13.The host cell of claim 12, wherein said animal cell is a human, mouse,rat, monkey, chicken, dog, cat, horse, pig, cow, sheep, goat or hamstercell.
 14. The host cell of claim 12, wherein said animal cell is a tumorcell.
 15. The host cell of claim 14, wherein said tumor cell is anovarian cancer or breast tumor cell.
 16. The host cell of claim 8,wherein said polynucleotide is introduced into said cell by means of avector.
 17. The host cell of claim 16, wherein said host cell expressessaid polynucleotide to produce a tumor suppressor polypeptide.
 18. Amethod of preparing a polypeptide, comprising the steps of: (a)providing a polynucleotide in accordance with claim 5; (b) introducingsaid vector into a host cell; (c) culturing said host cell underconditions effective to allow expression of the encoded polypeptide; and(d) collecting the polypeptide so expressed.
 19. An isolatedpolynucleotide fragment of SEQ ID NO:1 or SEQ ID NO:5 comprising atleast 100 continuous nucleotides thereof.
 20. The isolatedpolynucleotide fragment of claim 19, further defined as a polynucleotidefragment of SEQ ID NO:1 or SEQ ID NO:5 comprising at least 200contiguous nucleotides thereof.
 21. A nucleic acid detection kitcomprising, in suitable container means, the polynucleotide fragmentselected from the group consisting of the polynucleotide fragments ofclaims 19–20, and a detection reagent.
 22. A recombinant vectorcomprising the nucleotide fragment of claim
 19. 23. The recombinantvector of claim 22, further defined as comprising the nucleotidefragment of claim 20.